Cell suspensions

Br J Cancer . 2005 Jan 18; {Epub ahead of print}
Singlet oxygen luminescence as an in vivo photodynamic therapy dose metric: validation in normal mouse skin with topical amino-levulinic acid; Niedre MJ et al.; Although singlet oxygen ((1)O(2)) has long been proposed as the primary reactive oxygen species in photodynamic therapy (PDT), it has only recently been possible to detect it in biological systems by its luminescence at 1270 nm . Having previously demonstrated this in vitro and in vivo, we showed that cell survival was strongly correlated to the (1)O(2) luminescence in cell suspensions over a wide range of treatment parameters . Here, we extend this to test the hypothesis that the photobiological response in vivo is also correlated with (1)O(2) generation, independent of individual treatment parameters . The normal skin of SKH1-HR hairless mice was sensitised with 20% amino-levulinic acid-induced protoporophyrin IX and exposed to 5, 11, 22 or 50 J cm(-2) of pulsed 523 nm light at 50 mW cm(-2), or to 50 J cm(-2) at 15 or 150 mW cm(-2) . (1)O(2) luminescence was measured during treatment and the photodynamic response of the skin was scored daily for 2 weeks after treatment . As observed by other authors, a strong irradiance dependence of the PDT effect was observed . However, in all cases the responses increased with the (1)O(2) luminescence, independent of the irradiance, demonstrating for the first time in vivo an unequivocal mechanistic link between (1)O(2) generation and photobiological response.British Journal of Cancer advance online publication, 18 January 2005; doi:10.1038/sj.bjc.6602331 www.bjcancer.com.

Biochim Biophys Acta, 2005 Jan 18, 1721(1-3), 130 - 8 Epub 2004 Nov 11.
Boundary-element calculations for dielectric behavior of doublet-shaped cells; Sekine K et al.; In order to simulate dielectric relaxation spectra (DRS) of budding yeast cells (Saccharomyces cerevisiae) in suspension, the complex polarization factor (Clausius-Mossotti factor) beta for a single cell and the complex permittivity of a cell suspension epsilon(sus)* were calculated with a doublet-shaped model (model RD), in which two spheres were connected with a part of a ring torus, using the boundary element method . The beta values were represented by a diagonal tensor consisting of components beta(z) parallel to the rotation axis (z axis) and beta(h) in a plane (h plane) perpendicular to the axis . The epsilon(sus)* values were calculated from the complex permittivity of the suspending medium epsilon(a)* and the components of beta . The calculation was compared with that of a conventional prolate spheroid model (model CP) . It was found that model CP could be used as a first approximation to model RD . However, differences existed in beta(z) between models RD and CP; beta(z) showed three relaxation terms in the case of model RD in contrast with two terms in model CP . Narrowing the junction between the two spheres in model RD markedly decreased the characteristic frequency of one of the relaxation terms in beta(z) . This suggests that the structure of the junction can be estimated from DRS . Effects of the shape change from model RD to a two-sphere model (model RD without the junction) were also examined . The behavior of beta(z) in the two-sphere model, the relaxation intensity of which was much lower than model RD, was quite similar to that in a single-sphere model . These simulations were consistent with the experimental observations of the dielectric behavior of the yeast cells during cell cycle progression.

Vestn Ross Akad Med Nauk, 2004, (11), 8 - 17
{Results and outlooks of using cell technologies in the treatment of neurological diseases}; Cole electrical impedance model--a critique and an alternative; Department of Clinical and Biomedical Engineering, N-0027 Oslo, NorwayThe Cole single-dispersion impedance model is based upon a constant phase element (CPE), a conductance parameter as a dependent parameter and a characteristic time constant as an independent parameter . Usually however, the time constant of tissue or cell suspensions is conductance dependent, and so the Cole model is incompatible with general relaxation theory and not a model of first choice . An alternative model with conductance as a free parameter influencing the characteristic time constant of the biomaterial has been analyzed . With this free-conductance model it is possible to separately follow CPE and conductive processes, and the nominal time constant no longer corresponds to the apex of the circular arc in the complex plane.

Ann Urol (Paris), 2004 Dec, 38(6), 266 - 74
{Tissue engineering in urology}; Zini L et al.; Tissue engineering refers to the techniques that are aimed at regeneration of human tissues and organs . Two elements are necessary for these techniques: matrix and cells . Matrix is the scaffold where tissues may organise . Cells are either autologous cells stimulated to regenerate in vivo, aided by implantation of matrix ("guided tissue regeneration"), or autologous cells cultured outside the body (in vitro) and later returned as auto-transplants . All types of conventional tissue reconstructive surgery need tissue engineering . These techniques have been introduced recently into the clinical practice . One of the main limitations of reconstructive surgery in genitourinary tract is the lack of autologous tissue . Two autotransplants could be distinguished: coherent tissue structure or cell suspensions . The great number of studies published in this area emphasizes the importance of the future clinical implication in urology.

J Fluoresc, 2004 Nov, 14(6), 711 - 22
Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2; Paredes-Gamero EJ et al.; Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ({Ca2+}i) . In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in {Ca2+}i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380) . At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions . We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm) . ATP analogs: BzATP, ATPyS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2 . The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown . The P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease . The J774 cell line and mononuclear cells with a higher expression of P2X7 receptors show the same decrease in R340/380 as that induced by ATP . In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur . It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of {Ca2+}i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.

J Enzyme Inhib Med Chem, 2004 Oct, 19(5), 431 - 6
Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone; Mansour M et al.; Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases . Thymoquinone (TQ) has been proposed to be one of the major active components of the drug . Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells . TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min . Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions . In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4 . In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity . When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions . The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ . The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells . The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.

Dev Biol (Basel), 2000, 102, 125 - 9
The use of dimethylmethylene blue for virus photoinactivation of red cell suspensions; Wagner SJ et al.; Phenothiazine dyes and light have been known to have virucidal properties for over seventy years . This review will describe recent progress in the use of one phenothiazine dye, dimethylmethylene blue, for photo-inactivation of a number of RNA and DNA viruses in red cell suspensions under conditions that minimally affect red cell in vitro properties during 42-day 1-6 degrees C storage . Dimethylmethylene blue has a higher affinity for nucleic acid than the closely related phenothiazine, methylene blue . Virus photoinactivation appears to be mediated by singlet oxygen . The kinetics of photoinactivation depends on the virus studied, but for a given virus, is similar for both intracellular and extracellular forms . The similarity for inactivation of intracellular and extracellular virus suggests that a common target, such as nucleic acid, is involved . Finally, lymphocytes, which can harbour transfusion-associated viruses and can mediate transfusion-associated-graft-versus host disease, are sensitive to dimethylmethylene blue photoinactivation under virucidal conditions.

Cell Mol Biol Lett, 2004, 9(4B), 795 - 804
The cloning of sequences differentially transcribed during the induction of somatic embryogenesis in cucumber (Cucumis sativus L.); Linkiewicz A et al.; Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process . In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development . We identified and cloned 120 candidate cDNA fragments from differential display gels . The selected cDNAs were confirmed by reverse northern, and 83 were sequenced . The obtained sequences represent 64 independent transcripts . The search for similarities in the databases gave a significant result in 16 cases . The potential involvement of these sequences in somatic embryogenesis is discussed.

Neurosci Biobehav Rev, 2005 Jan, 28(8), 803 - 10
Transplantation of carotid body cells in the treatment of neurological disorders; Yu G et al.; Laboratory and clinical studies have shown that intracerebral transplantation of carotid body (CB) cells ameliorate Parkinsonian deficits . The recent clinical study by Arjona and colleagues indicated that CB autograft transplantation is a relatively simple, safe, and viable treatment for PD patients . In particular, Espejo and colleagues demonstrated that the therapeutic efficacy of intracerebral transplantation of the CB in PD was likely obtained through secretion of neurotrophic factors rather than the local release of dopamine, which suggests it possible and reasonable to extend the use of the CB as an efficacious graft source for neural transplantation . Thus, we transplanted CB cell suspensions into the ischemic penumbra within 1h after stroke surgery . The results revealed that CB transplantation also significantly reduced stroke-induced behavioral deficits and cerebral infarction . In this review, we focus on summarizing the physiological properties of the CB related to transplantation, describing briefly possible mechanisms responsible for the effect of CB transplantation, and introducing recent studies of the CB as a donor source for neural transplantation.

Chin Med J (Engl), 2005 Jan, 118(1), 6 - 11
Effects of antigen presentation of eosinophils on lung Th1/Th2 imbalance; Xie ZF et al.; BACKGROUND: Antigen-loaded eosinophils (EOSs) instilled intratracheally into mice were capable of inducing Th2-type cytokine production in the draining lymph nodes . The aim of the present study was to evaluate whether EOSs within the tracheobronchial lumen can stimulate Th2 cell expansion in the lung tissues . METHODS: Airway EOSs were recovered from ovalbumin-sensitized and -challenged BALB/c mice, these EOSs were then cocultured with CD4+ cells isolated from sensitized mice in the absence or presence of anti-CD80 or/and -CD86 monoclonal antibodies . Airway EOSs were instilled into the trachea of sensitized mice . At the day 3 thereafter, the lung tissues were removed and prepared into cell suspensions for culture . Cell-free culture supernatants were collected for detection of cytokines . RESULTS: Airway EOSs functioned as CD80- and CD86-dependent antigen-presenting cells to stimulate lung CD4+ lymphocytes to produce interleukin-4, interleukin-5 and interleukin-13, but not interferon-gamma in in vitro assay . When instilled intratracheally in sensitized recipient mice, airway EOSs primed lung Th2 cells in vivo for interleukin-4, interleukin-5 and interleukin-13, but not interferon-gamma, production during the in vitro culture that was also CD80- and CD86-dependent . CONCLUSION: EOSs within the lumina of airways could process inhaled antigen and function in vitro and in vivo as antigen-presenting cells to promote expansion of Th2 cells in the lungs.

Yi Chuan, 2004 Sep, 26(5), 676 - 82
{Differential Expression on Cell Wall Formative Genes in Cell Suspension Cultures of Populus tomentosa Carr.}; Li XG et al.; With fine and granular suspension cultures established from P . tomentosa, differential expression of 12 genes related to cell wall formation including cell expansion, cellulose synthesis, lignin synthesis, etc, were investigated by reverse transcript PCR (RT-PCR) technique . Fine suspension cells showed high expression on 3 alpha-Exp genes with the highest alpha-Exp2 followed byalpha-Exp3 and alpha-Exp1 suggesting their strong cell expansion, while granular suspension cells only exist high expression on alpha-Exp3 indicating their later period of cell expansion or initial stage of secondary cell wall formation . Among the 6 genes relating to cellulose synthesis, both fine and granular cultures have high expression on CesA4 and CaSy1 and some expression on CesA1bCesA3-1 and CesA3-2, indicating their primary cell wall formation initiated, however a little higher expression on CesA3-1 and CesA3-2 in granular cells may reflect its more cellulose synthesis and more primary cell wall formation . The occurrence of Lac1 and Unk1 could not been detected in the 2 cell suspension cultures, showing no significant lignin biosynthesis and matural secondary cell wall formation.

Neurotox Res, 2004 Dec, 7(1,2), 169 - 178
Treatment of spinal cord injury with co-grafts of genetically modified Schwann cells and fetal spinal cord cell suspension in the rat; Feng SQ et al.; Fetal spinal cord cells, Schwann cells and neurotrophins all have the capacity to promote repair of injured spinal cord in animal models . To explore the possibility of using these approaches to treat clinical patients, we have examined whether a combination of these protocols produces functional and anatomical improvement . The spinal cords of adult rats (n=16) were injured with a modified New York University (NYU) device (10 gram.5cm) . One week after injury, the injured cords were injected with Dulbecco-modified Eagles Medium (DMEM, control group), or fetal spinal cord cell suspension (FSCS) plus nerve growth factor (NGF) gene-modified Schwann cells (SC) and brain-derived neurotrophic factor (BDNF) gene-modified SC (treatment group) . The rats were subjected to BBB (Basso, Beattie, Bresnahan, Exp . Neurol . 139:244, 1996) behavioral tests . Anterograde tracing of corticospinal tract was performed before sacrifice 3 months after the treatment . The results showed that the combination treatment elicited a robust growth of corticospinal axons within and beyond the injury site . A dramatic functional recovery in the treatment group was observed compared with the control group . We conclude that the combination of FSCS with genetically modified Schwann cells over-expressing NGF and BDNF was an effective protocol for the treatment of severe spinal cord injury.

Vnitr Lek, 2000 Nov, 46(11), 785 - 93
{Will transplantation of peripheral blood stem haematopoietic cells replace definitely bone marrow transplantation?}; Mayer J; Findings assembled for a long time, practically from the end of the Second World War along with modern technology of genetic engineering which brought mass production of growth factors led at the beginning of the nineties of the 20th century to a rapid rise of transplantations of peripheral stem cells . Without exaggeration it may be said that the last decade of the 20th century is the decade of transplantations of peripheral stem cells . Peripheral stem cells comprise a wide range of haematopoietic cells incl . stem cells which are after a stimulus (antitumour chemotherapy or growth factors) released from bone marrow in high concentrations into the peripheral blood stream from where they can be obtained very simply by modern blood cell separators . They are suitable for autologous as well as allogenic transplantations . In autologous applications they have almost replaced bone marrow . Restoration of haematopoiesis is after their use more rapid as the transplant is richer . As regards allogenic application caution is still apparent, although even their ratio is very significant . However a higher rate of reactions of the graft to the host is feared . It seems however that this fear is not justified and that the richer transplant of peripheral stem cells can ensure a higher anti-tumourous effect of the transplantation . Peripheral stem cells are also used successfully in transplantations after so-called non-myeloablative regimes . It is beyond doubt that the use of peripheral stem cells will increase further . We may expect application of other growth factors with a higher mobilizing capacity . Peripheral stem cells will be subjected to further modifications in vitro . It will be also possible to increase their amount . A rich transplant makes it also possible to use it in transplantations from haploidentic donors . No doubt stem cell suspensions will become the objective of gene therapy.

Tree Physiol, 2005 Mar, 25(3), 277 - 88
Activation of stress-responsive mitogen-activated protein kinase pathways in hybrid poplar (Populus trichocarpa x Populus deltoides); Hamel LP et al.; Plant mitogen-activated protein kinase (MAPK) cascades are important amplifying modules that can rapidly transduce stress signals into various appropriate intracellular responses . Several extracellular regulated kinase (ERK)-type MAPKs involved in plant defense signaling have been identified in herbaceous species, but no MAPK cascade has yet been characterized in a tree species . We examined the signal transduction events that lead to activation of defense mechanisms in poplar, a major forest species of economic and ecological importance which is becoming the model tree system for studying stress and adaptation responses . We show that, in poplar cell suspensions and leaf tissue, chitosan, a non-host-specific elicitor, and ozone, a strong oxidant and atmospheric pollutant, induce rapid and transient activation of at least two myelin basic protein (MBP) kinases with apparent molecular masses of 44 and 47 kD . The chitosan- and ozone-activated kinases have characteristics of MAPKs-they preferentially phosphorylate MBP, require tyrosine and threonine phosphorylation to be activated and are specifically recognized by anti-ERK and anti-pERK antibodies . Moreover, activation of these poplar MAPKs by chitosan or ozone is dependent on the production of reactive oxygen species; the influx of calcium ions via membrane channels; the activation of an upstream, membrane-localized component; and a cognate MAPK kinase (MAPKK) . These data suggest that biotic and abiotic challenges activate MAPKs in poplar, as in herbaceous species, which then function as a convergence point for pathogen defense and oxidant stress signaling cascades.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 83 - 5
{Experimental research on immunological rejection of brain tissue allograft.}; Han Y et al.; AIM: To investigate the occurrence of rejection in brain tissue transplantation . METHODS: Ventral mesencephalon(VM) single cell suspensions from newborn rats were allografted into the striata of Parkinson's disease model recipient rats . Surviving animals were sacrificed at 2, 4 and 6 wk after transplantation, and the brain tissues were stained with HE or immunocytochemically to inspect the expression of tyrosine hydroxylase (TH) and major histocompatibility complex class II antigens (MHC II) . RESULTS: High MHC II expression was observed within and around the allografts compared with control at 2 wk after grafting . MHC II expression then decreased at 4 wk and at 6 wk only few immunopositive cells remained . The number of TH positive neurons was low at 2wk, but increased at 4 and 6 wk after transplantation . CONCLUSION: The brain is not an "immunologically privileged site" and graft rejection exists in brain tissue transplantation . This rejection maybe induced by MHC II . Therefore it is necessary to use immunosuppressants in brain transplantation.

Yonsei Med J, 2004 Dec 31, 45(6), 1025 - 34
A non-frozen living tissue bank for allotransplantation using green tea polyphenols; Hyon SH; Generally, mammalian cells and living tissues can be cryopreserved in a frozen state at very low temperatures over a long storage term . The survival rate of cell suspensions is often acceptable however, living tissues suffer a variety of injuries . In this paper, it was demonstrated that the addition of polyphenols extracted from green tea to conventional cell culture medium and tissue compatible liquid, can control cell proliferation and also preserve tissues for several months at ordinary room temperature, including such tissues as blood vessels, cartilage, islet cells and corneas . This protocol allows a non-frozen living tissue bank to be established using the preservation fluid described.

Exp Oncol, 2004 Dec, 26(4), 329 - 33
Production of nitric oxide by hypoxic radiosensitizer sanazole; Kondakova IV et al.; AIM: In this work we investigated the ability of hypoxia-selective radiosensitizer - sanazole to produce nitric oxide (NO) . METHODS: NO formation was determined by spectophotometric method in the reaction with sanazole and oxyhemoglobin . In suspensions of lymphoma EL-4 and mastocytoma P 8815 cell NO production was estimated indirectly as nitrite concentration in the supernatant fraction . RESULTS: Transformation of oxyhemoglobin by sanazole to methemoglobin suggested the dissociation of nitro group in aqueous solution and denitration of molecules . Addition of sanazole to hypoxic tumor cell suspension resulted in the increase of nitrite content in tissue culture medium . CONCLUSION: Presented data suggest the ability of sanazole to produce NO that may be important in a probable mechanism for antitumor and immunomodulating properties of this radiosensitizer.

Sci China C Life Sci, 2004 Oct, 47(5), 406 - 15
Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression; Wu X et al.; AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insect-baculovirus expression systems, each having different characteristics . AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect, A . californica, makes AcNPV less suitable for large scale protein synthesis . In contrast, BmNPV can only infect the silkworm, Bombyx mori, which is well-known for its easy rearing and large size . These characteristics make the BmNPV system especially suitable for large-scale industrial expression . To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV . The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV . Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF . This construct is able to express the bFGF protein both in silkworm larvae and in common-use cell lines, sf21, sf9 and High-five . Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases . This knockout mutation improves the production efficiency of the bFGF recombinant protein.

Leuk Lymphoma, 2005 Mar, 46(3), 303 - 11
The role of interleukin-3 in classical Hodgkin's disease; Aldinucci D et al.; Classical Hodgkin's disease (HD) is a peculiar form of lymphoma characterized by a low frequency of tumor cells, the so-called Hodgkin (H) and Reed/Sternberg (RS) cells, embedded in a background of non-neoplastic (reactive) cells believed to be recruited and activated by H - RS cell-derived cytokines/chemokines . How these tumor cells can survive in such a seemingly hostile environment has confused researchers . We have previously identified interleukin (IL)-3 receptor (R) expression as a common feature of classical HD and unveiled the potential role of IL-3 as a growth and anti-apoptotic factor for H - RS cells . More then 90% of malignant cells of classical HD usually express the alpha chain of the IL-3R (IL-3Ralpha), as evidenced by immunostaining of frozen sections and cell suspensions from neoplastic lymph nodes . Consistently, HD-derived cell lines (L428, KMH2, HDLM2 and L1236) express the alpha and beta chains that form IL-3R, both at the mRNA and protein level, with a molecular size of IL-3Ralpha identical (70 kDa) to that expressed by human myeloid cells . Exogenous IL-3 promotes the growth of cultured H - RS cells, such an effect being potentiated by IL-9 and stem cell factor (SCF) co-stimulation, and is able to partially rescue tumor cells from apoptosis induced by serum deprivation . Finally, cultured H - RS cells are able to increase the production of IL-3 by pre-activated T cells, suggesting an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms . This review will outline the biological activity of IL-3 and summarize the evidence indicating IL-3 as a growth and anti-apoptotic factor for H - RS cells in classical HD.

Growth Factors, 2004 Dec, 22(4), 281 - 9
The Secretion of Endothelin-1 by Microvascular Endothelial Cells from Human Benign Prostatic Hyperplasia is Inhibited by Vascular Endothelial Growth Factor; Stachon A et al.; Prostate growth seems to be influenced by paracrine factors like endothelin-1 (ET-1), originating from the microvascular endothelium . Recently, we reported on the first isolation and primary culture of microvascular endothelial cells (HPEC) derived from tissue of human benign prostatic hyperplasia (BPH) . Therefore, direct investigation of growth factor secretion by HPEC is now possible . BPH tissue was cut into small cubes and gently squeezed after incubation with dispase . HPEC were cultured from the resulting cell suspension after a stepwise selection by use of superparamagnetic beads coated with antibodies against endothelial specific antigens . HPEC were characterized by flow cytometry . After the incubation of HPEC either with vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-alpha), or adenosine triphosphate (ATP), the secretion of ET-1 was measured by ELISA . HPEC showed a typical endothelial morphology . They were positive for von Willebrand factor and CD31 . The ET-1 secretion of HPEC was inhibited by VEGF, but was unaffected by TNF-alpha or ATP . Furthermore, histochemistry revealed that in vivo microvascular endothelial cells were negative for ET-1 . Because of the suppression by the widespread VEGF, it is unlikely that ET-1 from the microvascular endothelium acts as a growth factor in human BPH.

Biotechnol Bioeng . 2004 Dec 22; {Epub ahead of print}
In situ extraction strategy affects benzophenanthridine alkaloid production fluxes in suspension cultures of Eschscholtzia californica; Klvana M et al.; The effect of contact between cells and extractive phase on secondary metabolite production was investigated in two-phase suspension cultures of Eschscholtzia californica . A system was designed to extract benzophenanthridine alkaloids from the cell culture, without contact between XAD-7 resins and the cells: only medium was recirculated through a column packed with the extractive phase . This strategy was compared to the classic method of addition of resins directly into the cell suspension . Removal of the product directly from the medium enabled important increases in production of alkaloids, namely a 20-fold increase in sanguinarine production and a 10-fold increase in chelerythrine, with high recovery in the resin . The recirculation strategy greatly simplified the production process since the resins are easily recovered from the cell culture and enable harvest of product without termination of culture . However, due to limited flow rate, the recirculation strategy was slightly less effective than direct addition of resins into the cell suspension . In addition to enabling increased production, removal of secondary metabolites from the medium changed metabolic flux distribution, testifying to a complex control mechanism of production . (c) 2004 Wiley Periodicals, Inc.

Lab Chip, 2005 Feb, 5(1), 64 - 73 Epub 2004 May 26.
Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel; Chang WC et al.; A basic step in many biological assays is separating and isolating different types of cells from raw samples . To better meet these requirements in microfluidic devices for miniature biomedical analytical systems, an alternative method for separating cells has been devised by mimicking the physiological process of leukocyte recruitment to blood vessel walls: adhesive cell rolling and transient tethering . Reproducing these interactions for cells on surfaces of microstructured fluidic channels can serve to capture and concentrate cells and even to fractionate different cell types from a continuously flowing sample . To demonstrate this principle, two designs for microstructured fluidic channels were fabricated: an array of Square pillars and another with slender, Offset pillars . These structures were coated with E-selectin IgG chimera and the interactions of HL-60 and U-937 cells with these structures were characterized . With inflow of fluidic cell suspensions, the structures were able to efficiently capture and arrest cells directly from the rapid free stream flow . After capture, cells transit through the channel in three phases: cell rolling, cell tethering, and transient re-suspension in free stream flow before re-capture . Under these interactions, captured cells were enriched several hundred-fold from the original concentration . Additionally, among collected cells, the difference in flow-driven, adhesion-mediated cell transit in the Square design suggested that the two cell types could at least be partially fractionated.

Br J Ophthalmol, 2005 Jan, 89(1), 102 - 6
Neural progenitor cells from postmortem adult human retina; Mayer EJ et al.; BACKGROUND: Given the presence of neural progenitor cells (NPC) in the retina of other species capable of differentiating into multiple neural components, the authors report the presence of NPC in the adult human retina . A resident population of NPC suggests that the retina may constitutively replace neurons, photoreceptors, and glia . METHODS: Adult human postmortem retinal explants and cell suspensions were used to generate cells in tissue culture that display the features of NPC . The phenotype of cells and differentiation into neurons was determined by immunocytochemistry . Dividing cells were labelled with 5-bromo-2-deoxyuridine (BrdU) and neurospheres were generated and passaged . RESULTS: Cells labelled with nestin, neurofilament M (NFM), rhodopsin, or glial fibrillary acidic protein (GFAP) grew out from explant cultures . BrdU labelling of these cells occurred only with basic fibroblast growth factor (FGF-2) . Dissociated retina and pars plana generated primary neurospheres . From primary neurospheres, NPC were passaged to generate secondary neurospheres, neurons, photoreceptors, and glia . BrdU labelling identified dividing cells from neurospheres that differentiated to express NFM and rhodopsin . CONCLUSION: The adult human retina contains NPC and may have the potential to replace neurons and photoreceptors . This has implications for the pathogenesis and treatment of retinal disorders and degenerations, including glaucoma, and those disorders associated with retinal scarring.

Cryobiology, 2004 Dec, 49(3), 272 - 85
Restoring the endothelium of cryopreserved arterial grafts: co-culture of venous and arterial endothelial cells; Pascual G et al.; The use of arterial homografts in clinical practice is becoming increasingly common, yet there is an urgent need to address one of the most well-established problems associated with their use: the loss of integrity of the endothelium following cryopreservation . The partial lack of endothelium causes contact between the extracellular matrix and blood flow, which, in turn, often gives rise to thrombosis and/or restenosis . Our objective was first to attempt to replace the arterial endothelial cells lost during the cryopreservation process by seeding autologous venous endothelial cells, and to evaluate the behaviour of venous and arterial endothelial cells in co-culture . The idea was to establish whether venous endothelial cells would be accepted by arterial endothelial cells and could therefore be used to restore the endothelial lining for the subsequent use of these vessels in in vivo grafting procedures . For the co-culture experiments, endothelial cells were obtained from the jugular vein and both iliac arteries of the minipig by treatment with 0.1% type I collagenase . The venous endothelial cells were fluorescently labelled with the membrane intercalating dye PKH26 . Equal numbers of venous and arterial endothelial cells were mixed and co-cultured for 24h, 48h or 4 days . Cell viability, determined by 2% trypan blue staining and the TUNEL method, was established before and after fluorescence labelling . Cellular activity was determined by estimating PGI2 levels in the cultures . The proliferation index was established by {H(3)}thymidine (1muCi/ml) in the cell culture medium . For the in vivo tests, 5cm length segments of minipig iliac artery were used to establish the groups: control (n=6), fresh arterial segments; group I (n=16), cryopreserved arterial segments and group II (n=16), cryopreserved arterial segments seeded with autologous venous endothelial cells . The cryopreserved vessels in group II were seeded by flooding with a labelled venous endothelial cell suspension . Once seeded, the arterial segments were included in an in vitro flow circuit . All the specimens were processed for fluorescence and light microscopy, and scanning electron microscopy . The denuded endothelial surface was determined in each group . Cell death was evaluated by the TUNEL method . We confirmed the existence of intercellular PECAM1-type junctions between venous (PKH26+) and arterial cells in co-culture and the functional activity of the cells . The cryopreserved arterial segments showed a well-preserved wall structure . However, different size areas of marked endothelial denudation were detected . After seeding with labelled cells (PKH26+), these denuded areas of the cryopreserved artery were entirely covered by fluorescent cells . After seeding, a drop in the proportion of damaged endothelial cells was recorded . Despite some loss of seeded cells after inclusion in the in vitro flow circuit, the endothelial cell count was not significantly different to those recorded for control, non-cryopreserved specimens . In conclusion arterial and venous endothelial cells growing in co-culture modify their behaviour to form multilayers . The two cell populations form normal PECAM1 junctions and preserve their functional properties . Seeding autologous venous endothelial cells on the luminal surface of cryopreserved arterial segments serves to restore the integrity of the endothelial layer.

Cryobiology, 2004 Dec, 49(3), 201 - 10
Parasite cryopreservation by vitrification; James ER; Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation . Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification . With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying . Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification . Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites . For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S . mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling ( approximately 5100 degrees Cmin(-1)) to -196 degrees C . This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degrees C . Samples are rapidly warmed/diluted ( approximately 14,000 degrees Cmin(-1)) to recover the organisms from liquid nitrogen storage . Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos . Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification . Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date . Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained . And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods . Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.

Dev Growth Differ, 2004 Dec, 46(6), 515 - 22
Cloning and characterization of the tomato karyopherin alpha1 gene promoter; Mizrachy L et al.; The karyopherin alpha1 (LeKAPalpha 1) gene of tomato (Lycopersicon esculentum) encodes a receptor involved in nuclear import . To analyze the expression pattern of this gene, a genomic clone containing its upstream region was isolated and sequenced . To study the promoter functionality, a 2170 bp fragment (LM1), was fused to glucuronidase (GUS) and introduced into petunia cells by particle bombardment . For further characterization of the promoter, one inverse and three deletion constructs were studied in cell suspension . To follow its expression in tobacco leaves, transgenic plants expressing GUS under the control of the LM1 promoter were made . Expression of LM1-GUS was largely restricted to actively growing leaf regions, suggesting possible involvement of active cell division and plant growth regulators in LeKAPalpha 1 expression.

Muscle Nerve . 2004 Dec 17; {Epub ahead of print}
Enhanced reinnervation after neurotization with Schwann cell transplantation; Fukuda A et al.; We investigated the feasibility of using Schwann cell transplantation to enhance reinnervation after direct nerve-to-muscle neurotization (NMN) . The denervated anterior tibial muscle was neurotized by tibial nerve implantation, and Schwann cell suspension (transplantation group) or an equivalent volume of culture medium (control group) was injected at the implantation site . In the control group, few axons invaded the muscle, demonstrating that skeletal muscle was poorly permissive to the advancement of axons . In the transplantation group, a large number of regenerating axons grew for a longer distance throughout the muscle, and reinnervated motor endplates were significantly more abundant . Enhanced reinnervation and functional recovery of the muscle in the transplantation group was confirmed by a significant increase in the compound muscle action potential and in muscle weight . These results suggest that intramuscular Schwann cell transplantation has potential as a cell therapy to improve functional recovery after NMN . Muscle Nerve, 2005.

Drug Metab Dispos, 2005 Jan, 33(1), 115 - 20
Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures; Griffin SJ et al.; Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan . The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes . Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches . Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures . Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M) . In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions . However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites . The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).

J Biotechnol, 2005 Jan 26, 115(2), 167 - 77
Cloning of Tetrahymena cells in a chemically defined medium is possible in the presence of surfactants or at reduced temperature; Hellung-Larsen P; When Tetrahymena cells are exposed to physical or chemical stress they may die . The effect of a given stress depends on the culture medium, the temperature, and the manipulation of the cells . Cells in broth-medium or buffer solution are more resistant than cells in chemically defined medium (CDM) . A type of physical stress is caused by the hydrodynamic properties at the constriction of the pipette tip . This type of stress may be reduced/abolished by use of tips with maximal area and smoothness at the constrictions, underwater delivery of cell suspensions combined with gentle mixing, by use of reduced temperatures, by avoidance of medium-air interfaces or by addition of surfactants . By adjustment of these parameters it is possible to clone single cells of different species of Tetrahymena in CDM . In the presence of surfactants, cells can be cloned even under harsh manipulation . In absence of surfactants, cells can be cloned at 15 degrees C using mild manipulations . Tetrahymena cells are independent of unspecific growth factors and they do not exert autocrine growth control . Pluronic does not bind to the cells with significant affinity . Chemical stress cannot be counteracted by surfactants . Pre-stress (heat) protects the cells from subsequent lethal heat stress.

Differentiation, 2004 Oct, 72(8), 419 - 33
Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion; Getsios S et al.; Abstract Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases . These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results . Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK) . By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts . The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures . Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets . Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures . These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes . Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.

Radiat Res, 2005 Jan, 163(1), 63 - 71
A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation; Lu F et al.; Lu, F . and Wong, C . S . A Clonogenic Survival Assay of Neural Stem Cells in Rat Spinal Cord after Exposure to Ionizing Radiation . Radiat . Res . 163, 63-71 (2005).Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS) . Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage . Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation . Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor . The survival of the proliferating cells was determined by their ability to form neurosphere colonies . The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating . Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16 . Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy . When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord . Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord . In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord . However, neural stem cells retain their pluripotent and self-renewing properties after irradiation . A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.

Planta . 2004 Dec 17; {Epub ahead of print}
Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re-programming in cell suspension cultures of the model legume Medicago truncatula; Suzuki H et al.; Exposure of cell suspension cultures of Medicago truncatula Gaerth . to methyl jasmonate (MeJA) resulted in up to 50-fold induction of transcripts encoding the key triterpene biosynthetic enzyme beta-amyrin synthase (betaAS; EC 5.4.99.-) . Transcripts reached maximum levels at 24 h post-elicitation with 0.5 mM MeJA . The entry point enzymes into the phenylpropanoid and flavonoid pathways, L: -phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and chalcone synthase (CHS; EC 2.3.1.74), respectively, were not induced by MeJA . In contrast, exposure of cells to yeast elicitor (YE) resulted in up to 45- and 14-fold induction of PAL and CHS transcripts, respectively, at only 2 h post-elicitation . betaAS transcripts were weakly induced at 12 h after exposure to YE . Over 30 different triterpene saponins were identified in the cultures, many of which were strongly induced by MeJA, but not by YE . In contrast, cinnamic acids, benzoic acids and isoflavone-derived compounds accumulated following exposure of cultures to YE, but few changes in phenylpropanoid levels were observed in response to MeJA . DNA microarray analysis confirmed the strong differential transcriptional re-programming of the cell cultures for multiple genes in the phenylpropanoid and triterpene pathways in response to MeJA and YE, and indicated different responses of individual members of gene families . This work establishes Medicago cell cultures as an excellent model for future genomics approaches to understand the regulation of legume secondary metabolism.

Biotechnol Lett, 2004 Nov, 26(22), 1745 - 8
Improvement of growth and camptothecin yield by altering nitrogen source supply in cell suspension cultures of Camptotheca acuminata; Pan XW et al.; Nitrate at 70 m gave the highest biomass of Camptotheca acuminata in suspension culture in MS medium, but a NH(4)(+)/NO(3)(-) molar ratio of 5:1 (giving a total of 40 m N) gave the maximum camptothecin yield . A two-stage flask culture system was established to improve culture efficiency; cell dry weight, camptothecin content and yield was increased by 30%, 280% and 340%, respectively when compared with those of control, reaching up to 36 g l(-1), 0.36 mg g(-1), and 12.8 mg l(-1), respectively.

Plant Mol Biol, 2004 Aug, 55(6), 797 - 805
Activation of the oxidative burst by yeast elicitor in Catharanthus roseus cells occurs independently of the activation of genes involved in alkaloid biosynthesis; Pauw B et al.; In Catharanthus roseus cell suspensions, expression of several terpenoid indole alkaloid (TIA) biosynthetic genes, including those encoding strictosidine synthase and tryptophan decarboxylase, is coordinately induced by fungal elicitors such as yeast extract (YE) . This induction is mediated by several signaling steps including the biosynthesis of jasmonic acid, and the activation of the jasmonic acid-responsive ORCA transcription factors . We investigated a possible role of reactive oxygen species (ROS) as a second messenger in this system . YE was shown to activate the production of ROS, which was dependent on protein phosphorylation and calcium influx . However, ROS generation was neither necessary for the induction of genes involved in TIA biosynthesis by YE nor by itself sufficient to induce these genes . Therefore, we conclude that activation of the oxidative burst by YE occurs independently of the activation of genes involved in TIA biosynthesis.

Biotechniques, 2004 Dec, 37(6), 970 - 5
Multichannel apparatus for parallel monitoring of light scattering in Dictyostelium discoideum cell suspensions; Lusche DF et al.; Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development . We describe a device to monitor these oscillations in several samples in parallel . The apparatus consists of a thermostated cuvette holder where up to eight cuvettes containing cell suspension are inserted . Cells are aerated and kept in suspension via an airlift . Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes . The resulting signal is digitized and recorded with a sampling rate of two measuring points/second . The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point . This represents an advantage over the conventional single cuvette approach, as oscillation characteristics themselves are developmentally regulated . Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples, the behavior of wild-type and several mutant strains can be compared under identical experimental conditions.

Proteomics . 2004 Dec 9; {Epub ahead of print}
Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: Identification by mass spectrometry and bioinformatics; Boudart G et al.; Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach . An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed . Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants . Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride . From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm . Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI . Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena . Ten proteinases were also found as well as six proteins with unknown functions . Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families . complement.

Glia . 2004 Dec 8; {Epub ahead of print}
Phagocytosis of O4(+) axonal fragments in vitro by p75(-) neonatal rat olfactory ensheathing cells; Wewetzer K et al.; Olfactory ensheathing cells (OECs) have gained wide interest because of their unique regeneration-promoting capacity . However, despite their frequent use in regeneration studies, the characterization of the cells has remained fragmentary . In the present study, we analyzed freshly dissociated neonatal rat OECs at the light and electron microscopic level and studied their fate in vitro using a novel two-step labeling protocol based on antibody internalization . We report the identification and characterization of two distinct OEC populations in situ and in primary cell suspensions that differed in number, p75 NGF receptor expression, and O4 immunoreactivity . The major OEC population in primary cells suspensions did not express p75 but stained positive for the glycolipid O4 (p75(-)/O4(+)) . During culturing, these cells upregulated p75 expression and lost O4 immunoreactivity . Conversely, the minor OEC population consisted of p75(+)/O4(-) OECs that maintained p75 expression in vitro . Interestingly, ultrastructural analysis revealed not only that O4 immunoreactivity of p75(-) OECs was, in fact, due to O4(+) axonal fragments adhering to the cell surface but also that p75(-) OECs rapidly phagocytosed these fragments in vitro . Taken together, the identification of two distinct OEC populations in the neonatal olfactory bulb that converge into single p75(+) phenotype in vitro is reported . The observation that upregulation of p75 receptor expression in vitro was only apparent in those OECs closely associated with O4(+) axonal processes may suggest that axonal signalling in vivo negatively regulates p75 receptor expression . The strong phagocytic activity of OECs in vitro may reflect one important aspect of their physiological function . (c) 2004 Wiley-Liss, Inc.

Ultrason Sonochem, 2005 Apr, 12(5), 353 - 7
Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by pre-sonication incubation or by high cell density; Feril LB Jr et al.; To identify the factors involved in the inhibition of ultrasound (US)-induced free radical production and cell killing by pre-sonication incubation or by high cell density, we used different densities of U937 cells, and with (up to 2 h) or without pre-sonication incubations, the cell suspensions were exposed to 1 MHz US (10% duty factor at 100 Hz pulse rate; intensities 0.1-0.5 W/cm(2) for 1 min) . The intensity 0.3 W/cm(2) was used for cell killing experiments and 0.5 W/cm(2) for free radical experiments . Free radical production was determined by electron paramagnetic resonance (EPR)-spin trapping with DMPO while cell killing was determined by assays for lysis, loss of cell viability, apoptosis and necrosis . The results show that at higher cell densities, CO(2) in the medium rapidly increased, with shorter pre-sonication incubation required to attain complete inhibition of both free radical production and cell killing . Cell killing at 0.3 W/cm(2) and free radical production at 0.5 W/cm(2) were both inhibited at 10 million cells/ml without incubation, and at 2 million cells/ml incubated for 2 h before sonication . Level of CO(2) alone could not account for the inhibition; consumption of gases in the medium is also considered in the inhibitory effect of pre-sonication, while suppression of cavitational activities due to the "viscosity effect" is considered a more important factor in the inhibition by high cell density.

Toxicol In Vitro, 2005 Feb, 19(1), 55 - 61
Effects of ozone on isolated peripheral blood mononuclear cells; Larini A et al.; We have investigated the release of cytokines from isolated peripheral human blood mononuclear cells (PBMC) exposed to various ozone concentrations for 10 min and the release of both proinflammatory and immunosuppressive cytokine after 24, 48 and 76 h incubation . Ozonation was performed by exposing for 10 min equal cell numbers and volumes of cell suspension to equal volumes of a gas mixture (1:1 ratio) composed of oxygen-ozone with precise ozone concentrations ranging from 1.0 up to 80 mug/ml (0.02 up to 1.68 mM) . Markers of oxidative stress showed a significant relationship between ozone doses and both lipid peroxidation and protein thiol groups content . With the exception of the lowest ozone concentration, the cytokine production of PBMC was depressed particularly at concentrations from 40 mug/ml upwards . There was no significant effect on IL-6 production between exposed or unexposed cells, up to 72 h of incubation . IL-4 production was markedly affected by ozone exposure, showing a marked decrease even at the lowest ozone concentration (2.5 mug/ml) already after 24 h incubation . On the other hand, production of IFN-gamma and TNF-alpha was slightly stimulated by the lowest ozone dose either at all times or only after 72 h incubation, respectively . Analysis of the proliferation index (PI) is consistent with these results showing that, while the lowest concentration stimulates it, progressively increasing O(3) concentrations inhibit the PI . These data show that there is a significant relationship between cytokine production and ozone concentrations and that PBMC are very sensitive to oxidation particularly in presence of serum with low antioxidant capacity.

Stem Cells, 2004, 22(7), 1239 - 45
Insulin-like growth factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration; Kofidis T et al.; Insulin-like growth factor-1 (IGF-1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage . Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF-1 might enhance cellular engraftment and host organ-specific differentiation after injection in the area of acute myocardial injury . In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF-1 to the injected cell population . Green fluorescent protein (GFP)-labeled sv129 ESCs (2.5 x 10(5)) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice . Recombinant mouse IGF-1 (25 ng) was added to the cell suspension prior to the injection (n = 5) . Echocardiography was performed before organ harvest 2 weeks later . The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated . Mice with LAD ligation only (n = 5) and ESC transfer without IGF-1 (n = 5) served as controls . ESCs formed viable grafts and improved cardiac function . Left ventricular wall thickness was higher in the IGF-1 group (p = .025) . There was a trend toward higher fractional shortening in the IGF-treated group . Histological analysis demonstrated that IGF-1 promoted expression of alpha-sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01) . IGF did not affect the cellular response to the donor cells or tumorigenicity . IGF-1 promotes expression of cardiomyocyte phenotype in ESCs in vivo . It should be considered as an adjuvant to cell transfer for myocardial restoration.

Am J Pathol, 2004 Dec, 165(6), 2167 - 75
Distinctive properties of human adult brain-derived myelin progenitor cells; Ruffini F et al.; We used expression of the ganglioside A2B5 to isolate putative myelin progenitor cells from adult human central nervous system parenchyma and compared their phenotypic (expression of myelin lineage molecules) and functional (survival, proliferation) properties with mature oligodendrocytes (OLGs) derived from the same adult material and with A2B5(+) cells isolated from human fetal brain . A2B5(+) cells represented 3 to 5% of the total cell suspension derived from adult specimens . Results of protein (immunostaining) and RNA (polymerase chain reaction) analyses indicated that the adult A2B5(+) cells were more committed to the OLG lineage than their fetal counterparts while continuing to retain properties of progenitor cells compared to the postmitotic mature OLGs . Although the adult A2B5(+) cells retained the capacity to divide, albeit at a reduced rate compared to fetal A2B5(+) cells, they showed reduced survival and process outgrowth compared not only to fetal cells but also to mature OLGs . Our results confirm the presence of progenitor cells committed to the OLG lineage in the adult human central nervous system but raise the issues regarding the intrinsic capacity of these cells to contribute to the process of remyelination that may be necessary during demyelinating diseases.

Biochem J . 2004 Dec 3; {Epub ahead of print}
Nitric oxide consumption through lipid peroxidation in brain cell suspensions and homogenates; Keynes RG et al.; Mechanisms inactivating nitric oxide (NO) are probably important in governing the physiological and pathological effects of this ubiquitous signalling molecule . Cells isolated from a brain region rich in the NO signalling pathway, the cerebellum, consume NO avidly . This property was preserved in brain homogenates and required both particulate and supernatant fractions . A purified fraction of the particulate component was rich in phospholipids and NO consumption was inhibited by procedures that inhibited lipid peroxidation, namely a transition metal chelator, the vitamin E analogue Trolox, and ascorbate oxidase . The requirement for supernatant was accounted for by its content of ascorbate, which catalyses metal-dependent lipid peroxidation . The NO-degrading activity of the homogenate was mimicked by a representative mixture of brain lipids together with ascorbate and, under these conditions, the lipids underwent peroxidation . In a suspension of cerebellar cells, there was a continuous low level of lipid peroxidation and consumption of NO by the cells was reduced ~50 % by lipid peroxidation inhibitors . Lipid peroxidation was also abolished when NO was supplied at a continuous low rate (~100 nM/min), which explains why NO consumption by this process is saturable . Part of the activity remaining after inhibition of lipid peroxidation was accounted for by contaminating red blood cells but there was also another component whose activity was greatly enhanced when the cells were maintained in air-equilibrated conditions . A similar NO-consuming process was present in cerebellar glial cells grown in tissue culture but not in blood platelets or white cells, suggesting a specialised mechanism.

Exp Clin Endocrinol Diabetes, 2004 Nov, 112(10), 580 - 6
Influence of hepatocyte-rich liver cell mixture and liver fibroblasts on prolonging graft islet survival in rats without immunosuppressive drugs; Soto-Montenegro L et al.; BACKGROUND: We noted that a liver cell suspension, made up of a mixture of several kinds of hepatic cells, affected allogenic islet survival when it was transplanted into the liver, mixed with the islets or separately . AIM: To study if this effect was related to a liver cell mixture rich in hepatocytes (hp) or to liver fibroblasts (fb) . METHODS: We studied 14 groups of rats: (A) a sham group with saline; (B) a group receiving transplantation with hepatic cells alone; (C) a control group, with islets alone via the portal vein, without hepatic cells (hp or fb) . For the other groups, we used a different ratio of cells/islets (100 : 1, 150 : 1 and 200 : 1) and different co-transplantation techniques with both types of cells . For the D, E, J groups, a mixture of hepatocytes (hepatocyte-rich liver cell mixture) or fibroblasts with islets was injected into the portal vein . For the other groups, we used a sequential procedure with a 15 minute interval between a first injection of hp or fb into the portal vein or into the vena cava, and a second injection of islets always into the portal vein; thus, it was a sequential portal/portal procedure with hepatocyte-rich liver cell mixture (hp) (F, G) or fibroblasts (K, L) and a sequential cava/portal with hp (H, I) or fibroblasts (M, N) . RESULTS: Most of the co-transplantation groups showed functional islets (blood glucose < 250 mg/dl) on the first or second day of transplantation; after several days they once again had high glucose levels, though not as high as pre-transplantation . There was statistical significance (p < 0.001) between the presence or not of hepatic cells to obtain prolongation of graft survival (blood glucose < 250 mg/dl) . Statistical significance (p < 0.001) was found for several sequential groups with hp (F, I) and fb (K, L) . It was also remarkable that 3 rats (37.5 %) from the I group (sequential cava/portal with hp/islets 200 : 1) were euglycemic (blood glucose < 150 mg/dl) for more than 3 months . ANOVA showed a large interaction between the type of transplant performed and the cellular ratio used, with a significance of p < 0.001 . Histological studies in rats with prolonged euglycemia, showed insulin-producing cell aggregates in the liver, while there was a remarkable decrease in insulin-producing cells in the remaining islets of pancreatic tissue . CONCLUSION: The results showed a marginal prolongation of islet graft survival when they are co-transplanted with a hepatocyte-rich liver cell mixture or with liver fibroblasts . The mechanism does not seem to be a cellular interaction between different hepatic cells and islets, but some kind of cellular interaction or released factor from either two cell types on the immune system, blocking or modulating it, at least temporarily.

Bioprocess Biosyst Eng . 2004 Dec 1; {Epub ahead of print}
Simple and rapid determination of metabolite content in plant cell culture medium using an FT-IR/ATR method; Hashimoto A et al.; A simple, rapid and accurate mid-infrared (MIR) spectroscopic method for simultaneously determining the product (ethanol) content and the nutrient (sugar) content in plant-cell culture media was developed using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory . We assessed the potential of this method by comparing it to a high-performance liquid chromatography (HPLC) method, and using the developed method to measure the ethanol and sugar contents simultaneously in liquid culture media with rice and tabacum cell suspensions, respectively . The experimental results suggest that the sugar consumption and ethanol production behaviors of the plant cell suspensions can be non-destructively and simultaneously monitored using the developed method . Furthermore, the spectroscopic method provided in this study could be developed into a technique that could be used to analyze the overall kinetics of the metabolism of the plant cell suspensions.

Am J Respir Cell Mol Biol . 2004 Dec 2; {Epub ahead of print}
Identification and characterisation of human pulmonary dendritic cells; Demedts IK et al.; Dendritic cells (DC) are specialized antigenpresenting cells, linking innate and adaptive immune responses, and thus play an important role in immunologically mediated diseases, including pulmonary diseases such as asthma and respiratory viral infections . While much is known about the characteristics of lung DC in animal models, very few data concerning human lung DC are available . The goal of our study was to identify and characterise dendritic cells in human lung by preparing single cell suspensions from surgical resection specimens and subsequent labeling with the recently developed BDCA (Blood Dendritic Cell Antigen) markers . A straightforward isolation procedure was developed to avoid phenotypical and functional changes induced by extensive purification methods . In this way, human lung DC were directly identified without the need for an additional adherence step for further purification . For the first time, we demonstrate the presence of three previously unidentified DC subsets in human lung digests: myeloid DC type 1 (BDCA1+/ HLA-DR+), myeloid DC type 2 (BDCA3+/ HLA-DR+) and plasmacytoid DC (BDCA2+/ CD123+) . The presence of CD1a+ DC in the human lung was confirmed . The identification and characterisation of different human pulmonary DC subtypes is of great importance for the future development of DC-based immunotherapies.

Biomaterials, 2005 May, 26(14), 2173 - 81
The enhancement of recombinant protein production by polymer nanospheres in cell suspension culture; Ryu JH et al.; Recombinant Chinese hamster ovary (rCHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins . Three-dimensional suspension culture is preferred to two-dimensional monolayer culture for the efficient large-scale culture of rCHO cells and subsequent mass production of recombinant proteins . Previously, we have demonstrated that the use of plain polymer nanospheres enhances the growth of anchorage-dependent animal cells (human embryonic kidney 293 cells) in suspension culture in serum-containing medium . Vitronectin and fibronectin were adsorbed onto poly(lactic-co-glycolic acid) (PLGA) nanospheres (696nm in average diameter) by immersing the nanospheres in fetal bovine serum . In this study, we investigated if the use of vitronectin/fibronectin-adsorbed polymer nanospheres enhances recombinant protein production in rCHO cell suspension culture in serum-free medium . Cell aggregate formation may be critical for the survival and growth of anchorage-dependent animal cells in suspension culture, and cells in single cell suspension may result in cell death . The addition of vitronectin/fibronectin-adsorbed nanospheres to rCHO cell suspension culture promoted the rate and efficiency of cell aggregate formation . The nanospheres enhanced cell growth (2.9 folds on day 10) and, importantly, recombinant antibody production (1.8 folds on day 14), compared to suspension culture without nanospheres . The viability of cells in the aggregates in the nanosphere-added culture was high for the entire culture period of 14 days . Apoptotic activity of cells was much lower in the nanosphere-added culture than in the culture without nanospheres on day 5 . The nanosphere suspension culture method developed in this study may be useful for the mass production of recombinant proteins through large-scale suspension culture of anchorage-dependent animal cells.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1666 - 73
Effect of subculture and elicitation on instability of taxol production in Taxus sp . suspension cultures; Kim BJ et al.; The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable . To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics . With Taxus subcultures we observe alternating states of high and low productivity . Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one . These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing . High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition . Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment . Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties . To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture . Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures . The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.

Front Biosci, 2005 Jan 1, 10, 367 - 78 Print 2005 Jan 1.
Managing chronic pain with encapsulated cell implants releasing catecholamines and endogenous opiods; Winn SR et al.; Spinal injections (intrathecal) of norepinephrine and/or opiod agonists are antinociceptive and when administered together may act in synergy . Spinal implants of adrenal chromaffin cells are an effective method for sustained delivery of the analgesic substances norepinephrine and enkephalin to the central nervous system (CNS) . One method of packaging and implanting cell-loaded devices into the intrathecal space of recipients is by encapsulating the cell suspensions in a polymer membrane prior to implantation . Cells/tissue packaged within an encapsulating membrane obviate the need for immunosuppressive therapies in transplant recipients . In addition, device output can be quantified prior to implantation, and following the removal of the spinal implant . The ability to retrieve the devices with the present tubular configuration also confers an additional margin of safety over unencapsulated chromaffin cell implants . This paper reviews the research and clinical observations of cellular transplants containing adrenal chromaffin cells for relieving chronic pain . Encapsulated cell technology is discussed with an emphasis on our experiences developing pain-modulating clinical devices . The human-sized prototype devices were loaded with enzymatically isolated bovine chromaffin cells and maintained in vitro for 7 - 8 days in serum-free media . Two days prior to implantation, each device was assayed by static incubation to measure catecholamine and met-enkephalin output, and qualified devices (n = 6) were implanted into the sheep subarachnoid space for 6 weeks . Following a 6 week in life period, the retrieval forces of prototype devices were measured during removal from the subarachnoid space . Static incubation of the devices immediately following retrieval and after a 24 hour re-incubation period were used to quantify norepinephrine and met-enkephalin secretion profiles . This study demonstrated the safety, retrievability and maintenance of pharmacologically active encapsulated chromaffin cell-loaded devices with human implant dimensions.

Dig Liver Dis, 2004 Nov, 36(11), 735 - 43
Optimisation of isolation of richly pure and homogeneous primary human colonic smooth muscle cells; Tattoli I et al.; BACKGROUND: Inherent properties of gastrointestinal smooth muscle can be assessed using isolated cell suspensions . Currently available isolation techniques, based on short 2-h enzymatic digestion, however, present the disadvantage of low cellular yield with brief viability . These features are an important limiting factor especially in studies in humans in which tissue may not be available daily and mixing of samples is not recommended . AIMS: To optimise the isolation procedure of cells from human colon to obtain a richly pure primary smooth muscle cell preparation . METHODS: Slices of circular muscle layer, obtained from surgical specimens of human colon, were incubated overnight in Dulbecco's modified eagle's medium supplemented with antibiotics, foetal bovine serum, an ATP-regenerating system and collagenase . On the following day, digested muscle strips were suspended in HEPES buffer, and spontaneously dissociated smooth muscle cells were harvested and used either immediately or maintained in suspension for up to 72 h . Cell yield, purity, viability, contractile responses, associated intracellular calcium signals and RNA and protein extraction were evaluated and compared to cell suspensions obtained with the current short digestion protocol . RESULTS AND CONCLUSION: The overnight isolation protocol offers the advantage of obtaining a pure, homogeneous, long-life viable cell suspension that maintains a fully differentiated smooth muscle phenotype unchanged for at least 72 h and that allows multiple functional/biochemical studies and efficient RNA extraction from a single human specimen.

Zentralbl Gynakol, 2004 Dec, 126(6), 373 - 7
{Investigations on regulation of HCG by cortisol (prednisolon) in trophoblast cells in vitro}; Hocker I et al.; OBJECTIVE: Trophoblast cells synthesize a variety of hormones, in which hCG plays a major role . On the strength of special enzymes they are capable of catalyzing the reaction cortisol <--> cortisone . In vitro experiments showed the influence on ACTH- and cortisol secretion by CRH, ACTH and prednisolon . In this study we describe the influence of cortisol (prednisolon) on hCG production of trophoblast cells in vitro . MATERIAL AND METHODS: Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step . After adjusting the cell suspension to a defined cell concentration of 1 x 10 (6) cells/ml cells were cultivated . The addition of prednisolon followed every eight hours . The samples were collected after 24 hours for a total of 96 hours also from unstimulated cultures . Culture supernatants were assayed for hCG by enzyme-immunometric methods . RESULTS: The addition of prednisolon (50 microg/ml) stimulates the concentration of hCG in a time-depending manner . CONCLUSIONS: The trophoblast cell shows an increase in the concentration of hCG after stimulation with cortisol . For the first time an influence of cortisol (prednisolon) on hCG production could be demonstrated in cultured trophoblast cells.

Brain Res, 2004 Dec 24, 1030(1), 94 - 102
Autologous transplantation of expanded neural precursor cells into the demyelinated monkey spinal cord; Oka S et al.; The objective of this study was to establish if neural precursor cells could safely be developed from biopsy of the subventricular zone (SVZ) in the non-human primate (marmoset), and to determine their myelinating potential after autologous transplantation into a demyelinated lesion . Small amounts of tissue were safely collected from the subventricular-subependymal zone of the adult primate brain under ultrasonography without any neurological deficit . Neural precursor cells were isolated and expanded in the presence of mitogen in vitro . The dorsal columns of the adult marmoset spinal cord were demyelinated by X-irradiation and intraspinal injections of ethidium bromide in the center of the radiation field . Cell suspensions of the neural precursors were microinjected through a micropipette into the demyelinated lesion site in the spinal cord . Lesions were histologically examined 3 weeks after transplantation . Light and electron microscopic examination of plastic embedded sections revealed a significant number of myelinating profiles in the transplantation zone; no myelination was observed in control lesions . The myelinated axons had predominantly peripheral patterns of myelination . These results demonstrate that autologous transplantation of neural precursor cells in the adult nonhuman primate can remyelinate demyelinated central nervous system (CNS) axons, thus suggesting the potential utility of such an approach in demyelinating lesions in humans.

Fitoterapia, 2004 Dec, 75(7-8), 696 - 701
Analysis of essential oils from wild and micropropagated plants of damiana (Turnera diffusa); Alcaraz-Melendez L et al.; Damiana is a medicinal plant with many traditional uses and a reputation as an aphrodisiac . Essential oils produced by this plant are used in traditional medicine, and for the preparation of liquors and tea . The composition of essential oils from wild damiana, plants grown with micropropagated methods involving cell suspension, and explants in solid medium, is presented . Relevant differences are observed in oils coming from wild and micropropagated plants, where micropropagated plants being more uniform with respect to quality and quantity . The most abundant constituents of the oils were caryophyllene oxide, caryophyllene, delta-cadinene, elemene and 1,8-cineol.

Clin Diagn Virol, 1995 Feb, 3(2), 139 - 53
CMV PCR monitoring in leucocytes of transplant patients; Ehrnst A et al.; Background: The presumed latency of cytomegalovirus (CMV) in leucocytes and the sensitivity of the polymerase chain reaction (PCR) raise a question of its clinical value . Objectives: To develop and standardize a CMV PCR as a diagnostic tool for CMV infection in solid organ and bone marrow transplant patients by comparing it to a likewise standardized isolation, rapid isolation and to clinical symptoms . Study design: The material comprised 822 EDTA peripheral blood samples from 96 solid organ and 119 bone marrow transplant patients . One sample from each of 21 healthy bone marrow donors and 25 blood donors were used as controls . Two million leucocytes were lysed and one-tenth of a volume was used in a nested PCR employing immediate early gene primers . Results: The limit of detection was approximately 10 gene copies of a CMV DNA clone and 1 TCID(50) of extracted DNA from a cell suspension . The specificity was >/=0.99 when tested in CMV seronegative individuals . The positive and negative predictive values were 0.62 and 1.00, respectively . When PCR was compared to virus isolation/rapid culture in individual patients, PCR was positive more frequently in solid organ transplant patients than was CMV isolation/rapid culture, but the difference was not significant in bone marrow transplant patients . In isolation-positive patients, PCR became positive in samples taken 1-2 weeks earlier . In 54 solid organ transplant patients with PCR-positive samples, CMV-associated symptoms were present in {Formula: see text} patients with CMV isolated from blood but in only {Formula: see text} patients without viraemia . In 17 bone marrow transplant patients treated with ganciclovir, PCR became negative during or immediately after treatment in {Formula: see text} (70%) episodes . This was true of {Formula: see text} (42%) solid organ transplant patients . Conclusion: Screening of transplant patients with CMV PCR can be standardized at a clinically relevant level so that antiviral therapy can be instituted early.

Ai Zheng, 2004 Nov, 23(11 Suppl 1), 1365 - 9
{Effect of Epidermal Growth Factor Receptor-selective Tyrosine Kinase Inhibitor Gefitinib on Nasopharyngeal Carcinoma Xenografts.}; Wang SS et al.; BACKGROUND & OBJECTIVE: Gefitinib,an anilinoquinazoline,is an orally active,selective epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor,which has been approved for the treatment of advanced non-small cell lung cancer . We have found that the proliferation of nasopharyngeal carcinoma (NPC) cell lines CNE1,CNE2,and SUNE1 was inhibited by Gefitinib.The present study was designed to evaluate the effect of Gefitinib alone or in combination with cisplatin (DDP) on NPC CNE2 xenografts . METHODS: Exponentially growing CNE2 cells were prepared into cell suspension (1x10(7) cells/ml) . Suspension of 200 mul of CNE2 cells was injected s.c . into the right flank area of the mice . After 7 days,when well-established tumors of 100-200 mm(3) were detected,mice were randomized into five groups: control group,Gefitinib (100 mg/kg) group,Gefitinib (200 mg/kg) group,DDP group,and Gefitinib (100 mg/kg) plus DDP group . Gefitinib was administered by oral gavage on days 1-5 of each week for 4 weeks . DDP was administered i.p . once a week for 4 weeks . Tumor volume was determined by direct measurement with caliper and calculated by the formula 1/2x(large diameter)x(small diameter)(2) . The mice were sacrificed at two days after the treatment ended; tumor masses were removed and weighed . The tumor inhibition rates were calculated . The student's test was used to evaluated the statistical significance of the results . RESULTS: Growth curves showed that tumor masses of control group grew more rapidly than ones of every treatment group . The average tumor volume was significantly smaller in Gefitinib (200 mg/kg) group than in control group (P=0.02) . The average tumor volume had no significant difference between Gefitinib (100 mg/kg) group and control group . The average tumor volume of DDP or Gefitinib (100 mg/kg) plus DDP group was smaller than that of control group(P=0.007 and 0.001,respectively) . The average tumor volume had no significant difference between DDP and Gefitinib (100 mg/kg) in combination with DDP group . The tumor inhibition rates of Gefitinib (100 mg/kg) group,Gefitinib (200 mg/kg) group,DDP group,and Gefitinib (100 mg/kg) plus DDP group were 26.3%,30.6%,45.7% and 54.8%,respectively . The average tumor weight after treatment had no significant difference between Gefitinib (100 mg/kg) group and control group.The average tumor weights of Gefitinib (200 mg/kg) group,DDP group,Gefitinib (100 mg/kg) plus DDP group were all smaller than that of control group . The average tumor weight had no significant difference between DDP group and Gefitinib (100 mg/kg) plus DDP group . CONCLUSION: Gefitinib could inhibit the growth of NPC CNE2 xenografts . Gefitinib in combination with DDP did not significantly potentiate the effect of DDP on NPC CNE2 xenografts.

Plant Cell Physiol, 2004 Oct, 45(10), 1413 - 25
Transgenic tobacco (Nicotiana tabacum L.) plants with increased expression levels of mitochondrial NADP+-dependent isocitrate dehydrogenase: evidence implicating this enzyme in the redox activation of the alternative oxidase; Gray GR et al.; Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction . One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 1.1.1.42), although the precise role of this enzyme is not yet known . Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX) . We have partially purified mtICDH from tobacco (Nicotiana tabacum L . cv . Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS-PAGE, which was verified by peptide sequencing . In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak . Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity . In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type . When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line . These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX.

Mol Cancer Res, 2004 Nov, 2(11), 606 - 19
HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization; Dragowska WH et al.; The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated . These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice . LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)) . Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors . Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors . Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015) . Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors . Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion) . These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.

Phytochemistry, 2004 Dec, 65(24), 3199 - 209
Oxidation of 8'-hydroxy abscisic acid in Black Mexican Sweet maize cell suspension cultures; Zaharia LI et al.; In a biotransformation study to prepare deuterium labelled phaseic acid (PA) from deuterated abscisic acid (ABA), the product contained fewer deuterium atoms than expected . Thus, spectroscopic data of isolated deuterated PA prepared from biotransformation of (+)-5,8',8',8'-d4-ABA in maize (Zea mays L . cv . Black Mexican Sweet) cell suspension cultures showed 83% deuterium incorporation at the 8'-exo position . Also, metabolism studies of (+)-4,5-d2-ABA in maize resulted in the isolation of deuterium labelled ABA derivatives, namely PA, dihydrophaseic acid (DPA), 4'-O-beta-D-glucopyranosylDPA, 8'-hydroxyPA, 8'-hydroxyDPA and 8'-oxoDPA, as deduced from spectroscopic methods . These combined results suggested the presence of an aldehyde intermediate which is either: (a) reduced to 8'-hydroxyABA and cyclized to PA, or (b) is hydrated and cyclized to 8'-hydroxyPA or (c) is further oxidized to the acid and cyclized to 8'-oxoPA . The chemical synthesis of this intermediate, as well as its biotransformation in maize cell cultures is presented.

Luminescence, 2004 Nov-Dec, 19(6), 319 - 21
Chemiluminescent analysis of the antioxidant and immunomodulation effects of several psychotropic drugs on peritoneal macrophages; Hadjimitova V et al.; The present study describes the application of several chemiluminescent (CL) methods for evaluation of antioxidant and immunomodulation effects of psychotropic drugs upon phagocytes: KO2-induced luminal-dependent CL for detection of superoxide anion radicals in a pure chemical system; PMA- and A23187-induced CL of peritoneal macrophages for detection of free radicals in cell suspension; and CL, produced by the luciferase-catalyzed luciferin + ATP reaction, for evaluation of cell viability before and after drug application . These methods provide also a way to investigate the location of drug action . It was found that the psychotropic drugs in fluence the 'oxidative burst' of macrophages through two mechanisms: by expression of drug antioxidant properties and/or by a direct immunomodulation effect.

J Exp Bot . 2004 Nov 22; {Epub ahead of print}
Reduction of cell size induced by enod40 in Arabidopsis thaliana; Guzzo F et al.; An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter . All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells . Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size . The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides . As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 760 - 3
{Study of immune responses induced by human papillomavirus type 18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice.}; Li A et al.; AIM: To examine the humoral and cellular immunoresponses induced by HPV18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice . METHODS: 54 BALB/c mice were divided into 9 groups randomly, and then vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immune adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) . After the third inoculation, blood samples were taken to measure specific antibody, and footpad swelling test was used to detect delayed-type hypersensitivity(DTH).Mice were killed and spleens were taken to prepare single spleen cell suspension for lymphocyte proliferation assay and CD4(+)/CD8(+), IFN-gamma(+) or IL-4(+) T cells double staining FACS assay . And then the pathological changes of the swollen footpad were observed . RESULTS: Compared with control, significant immunoresponses were observed in different experimental groups . The level of specific serum IgG against HPV in experiment groups was much higher than that of control group, and intramuscular immunization group had the highest antibody level . The footpad from immunized mice injected with virus-like particles was swollen and harden, and a large amounts of mononuclear cells can be seen in the footpad tissues . Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8(+) IFN-gamma(+) cell number, while CD4(+) IL-4(+) cell number was higher in intranasal immunization groups . The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immune responses . CONCLUSION: The recombinant plasmids could induce strong humoral and cellular immunoresponses in mice . Costimulatory molecule B7-2 could enhance the immune responses against HPV18 induced by the L1-E6 and L1-E7 DNA vaccines when used as an adjuvant.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 678 - 81
{Effect of tryptase inhibitors on histamine release from human colon mast cells.}; Xie H et al.; AIM: To investigate the effect of tryptase inhibitors (TPIs) on histamine release from human colon mast cells . METHODS: Human mast cells were prepared by digestion of colon tissue and with collagenase and hyaluronidase, cultured with four kinds of TPIs, leupeptin, protamine, TLCK, and lactoferrin for 15 min at 37degrees Celsius respectively . A glass fibre-based fluorometry assay was used to detect histamine in mast cell suspension . RESULTS: 200 mmol/L leupeptin and 100 mmol/L protamine were able to stimulate histamine release from colon mast cells, while TLCK and lactoferrin did not . All TPIs inhibited anti-IgE-induced histamine release in a concentration dependent manner, and the inhibitory rates were 48.7%, 36.7%, 40.2% and 34.1%, respectively . However preincubation of TPIs with mast cells for 20 min at 37degrees Celsius before adding anti-IgE had little effect on anti-IgE induced histamine release . All TPIs were able to inhibit calcium ionophore (CI)-induced histamine release, and the maximum inhibition rate was between 25%-32% . Inhibition on histamine release by leupeptin and TLCK obviously enhanced when colon mast cells were preincubated with them for 20 min before adding CI . However, under the same condition, protamine failed to inhibit histamine release . CONCLUSION: We prove for the first time that TPIs inhibit anti-IgE-and CI-induced histamine release from human colon mast cells, suggesting that it is possible to treat inflammatory bowel disease or other mast cell-related diseases by using TPIs.

Histochem Cell Biol . 2004 Nov 18; {Epub ahead of print}
Production and characterisation of cell- and tissue-specific monoclonal antibodies for the flatworm Macrostomum sp; Ladurner P et al.; Monoclonal antibodies (mABs) against various cell types of the basal free-living flatworm Macrostomum sp . were produced by immunising Balb/c mice with cell suspensions of disintegrated animals . We identified 360 positive supernatants with specific staining of various tissues, cell types, patterns or structures . Here we report immunocytochemical characterisation, histological stainings and isotyping of 11 mABs specific for muscle cells (MMu-1, MMu-2, MMu-3, MMu-4), digestive and prostate glands (MDr-1 and MDr-2, MPr-1), epidermal cells (MEp-1), the ventral nerve cord including neuron clusters (MNv-1), gastrodermal cells (MDa-1) and spermatids (MSp-1) . Confocal microscopy, histological techniques, electron microscopy and immunoblotting were applied to demonstrate stainings in juveniles, adults, starved or well-fed animals . Considering the current lack of specific markers the obtained mABs will be particularly helpful studying embryonic and postembryonic development, pattern formation, cell differentiation, regeneration and reproductive allocation in Macrostomum sp., and possibly other basal flatworms . The small size, ease of culturing, short generation time, transparency and the basal phylogenetic position specify Macrostomum sp . as a suitable model organism for comparative analyses within Platyhelminthes and to Drosophila and C . elegans.

Arch Dermatol Res, 2005 Jan, 296(7), 339 - 42 Epub 2004 Nov 18.
Neutrophils infiltrating ultraviolet B-irradiated normal human skin display high IL-10 expression; Piskin G et al.; Exposure to an erythemal dose of ultraviolet B (UVB) is known to induce interleukin (IL-10) expression in human skin . It is generally believed that this IL-10 is predominantly expressed by CD11b(+)HLA-DR(+) macrophages that infiltrate the UVB-exposed skin . This cytokine is presumed to contribute to the immunosuppressive effects of UVB by inhibiting cell-mediated immune responses . We recently demonstrated that neutrophils, which also invade UVB-irradiated skin, express CD11b and HLA-DR as well . In addition, we showed that the presence of these neutrophils affects T-cell responses in primary T-cell cultures derived from UVB-exposed skin . Since neutrophils invade UVB-exposed skin and, like macrophages, express CD11b and HLA-DR, we sought to determine whether neutrophils represent another source of IL-10 . Skin biopsies were obtained from four healthy volunteers before and 2 days after exposure to four minimal erythema doses of UVB . A series of immunohistochemical double-staining procedures using the following markers was performed: IL-10, CD11b, HLA-DR, CD36, neutrophil elastase, and CD66b . As expected IL-10 could be detected in CD11b(+)HLA-DR(+)CD36(+) macrophages in the epidermis and dermis of UVB-exposed skin . Surprisingly, the majority of the abundant IL-10 expression was found in CD11b(+)HLA-DR(+)elastase(+)CD66b(+) neutrophils . Cytospin preparations from dermal cell suspensions confirmed the IL-10 expression by neutrophils displaying characteristic multilobular nuclei . Thus, neutrophils in UVB-exposed skin express IL-10 and should be recognized as active coplayers in the creation of the UVB-induced immunosuppressive microenvironment.

Br J Oral Maxillofac Surg, 2004 Dec, 42(6), 532 - 7
Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits; Chen F et al.; To find out if it was possible to prefabricate bone graft in the shape of a human mandibular ramus that possessed a pedicle that carried blood . The pore size of the natural coral was about 200 microm with a porosity of about 36% . The natural coral was made into the shape of a human mandibular ramus . Marrow-derived osteoblasts were seeded into porous natural coral scaffolds in a density of 2 x 10(8)/mL in 300 microL cell suspension . After two days of in vitro incubation, five cell-coral complexes were implanted into cell donor rabbits under the inferior epigastric blood vessels to prefabricate a vascularised bone graft of specific shape . Two months later the bone formation was observed by gross inspection and histological examination . Two months after operation, a well-vascularised bone graft in the shape of the initial coral scaffold and with a blood-carrying pedicle had been regenerated successfully . Osteogenesis followed the pattern of endochondral bone formation . New bone could be seen on the surface and in the pores of coral on histological examination . We have shown that it is possible to fabricate vascularised bone graft in a predetermined shape using tissue engineering . This kind of bone graft may have future clinical application.

Mol Cell Biochem, 2004 Oct, 265(1-2), 19 - 26
Inhibition of Helicobacter pylori in vitro by various berry extracts, with enhanced susceptibility to clarithromycin; Chatterjee A et al.; The objective of this study was to evaluate the effects of various berry extracts, with and without clarithromycin on Helicobacter pylori . Resistance to clarithromycin by H . pylori has been reported, leading to interest in alternatives/adjuncts to therapy with clarithromycin . H . pylori American type culture collection (ATCC) strain 49503 was grown, cell suspensions were made in PBS and diluted 10-fold . One hundred microL of the suspension was then incubated for 18 h with extracts of raspberry, strawberry, cranberry, elderberry, blueberry, bilberry, and OptiBerry, a blend of the six berries, at 0.25-1% concentrations . Serially diluted cell suspensions were exposed for 1 h to clarithromycin at 15 microg/ml . Ten microl of bacterial samples from the 10(-7) dilution tube were plated and incubated for 18 h and the number of colonies were counted . Growth of H . pylori was confirmed by the CLO test . All berry extracts significantly (p < 0.05) inhibited H . pylori, compared with controls, and also increased susceptibility of H . pylori to clarithromycin, with OptiBerry demonstrating maximal effects.

Pigment Cell Res, 2004 Dec, 17(6), 627 - 35
The melanocortin receptor-1 gene but not the proopiomelanocortin gene is expressed in melanoblasts and contributes their differentiation in the mouse skin; Hirobe T et al.; Alpha-melanocyte stimulating hormone (alpha-MSH) added to serum-free primary culture of melanoblasts derived from epidermal cell suspensions of 0.5 d old C57BL/10JHir mice induced their differentiation . Analysis using the reverse transcription-polymerase chain reaction showed that the expression of the melanocyte-specific alpha-MSH receptor gene, melanocortin receptor-1 (MC1-R), had already been initiated before addition of alpha-MSH, and, in addition, no up-regulation of the MC1-R gene was observed after addition of alpha-MSH . However, no expression of the proopiomelanocortin (POMC) gene was observed before or after the addition of alpha-MSH . The expression of the MC1-R and POMC genes in the epidermis and dermis of the dorsal skin was surveyed from 13 d old embryos to 5.5 d old neonates . The expression of the MC1-R gene was first observed in the epidermis of 13 d old embryos, and gradually increased up to 0.5 d after birth, and thereafter remained constant . By contrast, the expression of the MC1-R gene in the dermis was first observed in 16 d old embryos, and gradually increased up to 3.5 d after birth, and thereafter remained constant . However, no expression of the POMC gene was observed in the epidermis or dermis of the dorsal skin at any age of mice tested . These results suggest that the expression of the MC1-R gene, but not of the POMC gene, plays an important role in the regulation of melanocyte differentiation in mouse skin.

J Nutr Biochem, 1999 Apr, 10(4), 205 - 9
A method for the determination of glucose synthesis in isolated bovine hepatocytes; Azain MJ et al.; A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented . This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods . Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with (14)C-labeled lactate or propionate as gluconeogenic substrates . Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin . Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting . Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant . Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose . This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.

Photochem Photobiol . 2004 Aug 1; {Epub ahead of print}
Endogenous Fluorescence Spectroscopy of Cell Suspensions for Chemopreventive Drug Monitoring; Kirkpatrick ND et al.; Cancer chemopreventive agents such as N-4-(hydroxyphenyl) retinamide (4HPR) are thought to prevent cancers by suppressing growth or inducing apoptosis in pre-cancerous cells . Mechanisms by which these drugs affect cells are often not known and the ability to monitor their effects is not available . In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment with 4HPR in ovarian and bladder cancer cell lines . Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity through redox status and protein content . Cells were treated with varying concentrations of 4HPR and measured in a stable environment with a sensitive fluorescence spectrometer . Results suggest that redox signal of all cells changed in a similar dose dependant manner but started at different baseline levels . Redox signal changes depended primarily on changes consistent with NADH fluorescence while the FAD fluorescence remained relatively constant . Similarly, tryptophan fluorescence decreased with increased drug treatment suggesting a decrease in protein production . Given that each cell line has been shown to have a different apoptotic response to 4HPR, fluorescence redox values along with changes in tryptophan fluorescence may be a response as well as an endpoint marker for chemopreventive drugs.

Clin Cancer Res, 2004 Nov 1, 10(21), 7171 - 8
Angiogenic profile of breast carcinoma determines leukocyte infiltration; Bouma-ter Steege JC et al.; To study the relationship between the angiogenic profile and leukocyte infiltration of tumors, single cell suspensions of archival frozen medullary and ductal breast cancer tissues were analyzed by flow cytometry . The amount of leukocytes and endothelial cells was measured, as well as the expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell fraction . A significantly higher number (3.2-fold) of infiltrating leukocytes was observed in medullary carcinoma . The composition of this infiltrate was similar to that seen in ductal carcinomas . The more intense infiltrate was explained by the approximately 3-fold enhanced endothelial ICAM-1 expression in medullary carcinoma . The angiogenic profile of all tumors was assessed by quantitative real-time reverse transcription-PCR analysis . Vascular endothelial growth factor (VEGF)-C and VEGF-D, but not VEGF-A, basic fibroblast growth factor, placental growth factor, and angiopoietins 1, 2, and 3 showed a relatively higher level of expression in ductal carcinoma than in medullary carcinoma . In vitro, both VEGF-C and VEGF-D were found to decrease endothelial ICAM-1 expression in the presence of basic fibroblast growth factor . These data suggest that in vivo angiogenic stimuli prevent the formation of an effective leukocyte infiltrate in tumors by suppressing endothelial ICAM-1 expression.

FEBS Lett, 2004 Nov 5, 577(1-2), 175 - 80
Double transfection improves small interfering RNA-induced thrombin receptor (PAR-1) gene silencing in DU 145 prostate cancer cells; Liu J et al.; The efficiency of small interfering RNA (siRNA)-induced gene knockdown is hampered by low transfection efficiency . We established a novel and simple double transfection method using specific siRNA duplexes targeted against human thrombin receptor PAR-1 in DU 145 prostate cancer cells . The initial siRNA transfection of cell suspensions followed by re-transfection of adherent cells on the following day resulted in undetectable PAR-1 mRNA and absent receptor protein . PAR-1 mRNA expression was silenced for up to five days . Functional studies showed that PAR-1 gene silencing in DU 145 cells abolished the modulating effects of thrombin on cell adhesion to the extracellular matrix proteins, fibronectin and laminin, thus demonstrating the essential role of PAR-1 in mediating thrombin effects on DU 145 cell adhesion.

Neurosci Lett, 2004 Nov 23, 371(2-3), 249 - 54
Neural progenitor cells do not differentiate prematurely in presenilin-1 null mutant mice; Wen PH et al.; Mice with a null mutation of the presenilin-1 (PS1-/-) gene die during late intrauterine life or shortly after birth and exhibit defects in cortical development . A previous report suggested that neurons differentiate prematurely in PS1-/- brain {M . Handler, X . Yang, J . Shen, Presenilin-1 regulates neuronal differentiation during neurogenesis, Development 127 (2000) 2593-2606} . Here we reexamined the issue of whether premature neuronal differentiation occurs in PS1-/- brain using fresh cell suspensions from embryonic E11.5 and E13.5 telencephalon where individual cell phenotypes can be easily determined with cell type specific markers . Immunostaining with seven neuronal specific markers (MAP2, beta-III tubulin, GABA, reelin, GluR2/3, calbindin, and calretinin) failed to reveal any evidence of premature neuronal differentiation in PS1-/- telencephalon . We also determined the fraction of cells expressing the neural progenitor marker nestin and found no evidence for premature depletion of neural progenitor cells in PS1-/- telencephalon . Moreover, based on MAP2 staining of tissue sections from E12.5 embryos the topography of newly generated neurons also appeared to be undisturbed in the telencephalon of PS1-/- embryos . These studies thus argue that premature neuronal differentiation is unlikely to be a core pathophysiological feature underlying the aberrant cortical development that occurs in PS1-/- brain.

J Biomech, 2005 Jan, 38(1), 117 - 24
On the mechanism of cell lysis by deformation; Takamatsu H et al.; In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates . The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates . The viability declines with decreasing gap size in the following order: 700 mM >150 mM >75 mM NaCl . This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size . The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration . In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton.

Anticancer Res, 2004 Sep-Oct, 24(5A), 2975 - 83
Applicability of the 2-nitroimidazole-sodium borocaptate-10B conjugate, TX-2060, as a 10B-carrier in boron neutron capture therapy; Masunaga S et al.; BACKGROUND: It is difficult to deliver a therapeutic amount of 10B from conventional 10B-carriers for boron neutron capture therapy (BNCT) throughout the target tumors, especially into the intratumor hypoxic cells which have low uptake capacities . We evaluated the usefulness of 5 new 10B-compounds (TX-2041, TX-2042, TX-2058, TX-2059 and TX-2060) as 10B-carriers in BNCT . They are 2-nitroimidazole-sodium borocaptate-10B (BSH) conjugates, that is, hybrid compounds that have both a hypoxic tumor cell sensitizing unit under gamma-ray irradiation, 2-nitroimidazoles and a thermal neutron-sensitizing unit, BSH . MATERIALS AND METHODS: The 5 new compounds were administered to SCC VII tumor-bearing mice intraperitoneally . As a control, BSH was also administered in the same manner . Then, the 10B concentrations in the tumors and normal tissues were measured by gamma-ray spectrometry . Based on the data of the pharmacokinetics analyses, TX-2060 was chosen for a subsequent tumor-irradiation study . SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, then treated with TX-2060 or BSH in the same manner as in the pharmacokinetics analyses . To obtain similar intratumor 10B concentrations during radiation exposure, irradiation with thermal neutrons or gamma-rays was started from 60 min after administration of the 10B-carrier . Right after irradiation, the tumors were excised, minced and trypsinized . The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU-labelling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . Meanwhile, the MN frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU . The clonogenic cell survival was also determined in mice given no BrdU . RESULTS: 10B distribution analyses in tumors, muscles, blood and liver indicated that TX-2060 has the most favorable characteristics for concentrating a sufficient amount of 10B in tumors and maintaining a high enough 10B concentration during irradiation . In addition, TX-2060 had a significantly stronger radio-sensitization effect with reactor thermal neutron beams than BSH on both total and Q cells in solid tumors . Further, TX-2060 clearly exhibited a radio-sensitization effect with gamma-rays, not only on total cells but also on Q and hypoxic tumor cells, which was not achieved by BSH . CONCLUSION: 10B-carrier, with a gamma-ray-sensitizing effect on tumor cells as well as the potential to keep 10B in tumors and sensitize tumor cells more markedly than conventional 10B-carriers, such as TX-2060, is a promising candidate for use in BNCT.

Z Rheumatol, 2004 Oct, 63(5), 385 - 92
{Mid-term results of autologous chondrocyte transplantation in knee and ankle . A one- to six-year follow-up study}; Dorotka R et al.; BACKGROUND: The reimplantation of autologous chondrocytes is a new technique in reconstruction of cartilage defects; initial results achieved with this technique have been promising . In an arthroscopic procedure, scales of cartilage are obtained from intact cartilage . The chondrocytes are then multiplied in special laboratories . A few weeks later, in a second procedure, the cartilage defect is filled with the cell suspension and closed with a flap of periosteum . METHOD: At our department, autologous chondrocyte transplantation (ACT) has been used in 10 patients since 1996, in 6 cases in the knee joint, and in 4 cases in the ankle joint . The mean age of the patients was 30 years . The mean size of the defect was 4 cm(2) . In 4 patients, a parallel surgical procedure was required at the time of removal . RESULTS: The mean duration of follow-up was 21/2 years . Six patients had good to excellent results, 3 patients had moderate results, and one patient a poor result . The modified Cincinnati rating scale was improved from 2.4 to 7.1 points, and the Lysholm score from 59.2 to 86.6 points . The AOFAS score for ankle joints had improved from 33 to 76 . CONCLUSION: We were able to show that ACT achieves improvement in the knee as well as ankle joint in the majority of patients . ACT appears to be a promising therapeutic concept for both joints.

Biotechnol Bioeng, 2004 Dec 30, 88(7), 880 - 9
Murine leukemia virus clearance by flocculation and microfiltration; Akeprathumchai S et al.; Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated . Murine leukemia virus is a retrovirus that is recommended by the U.S . Food and Drug Administration for validating clearance of retrovirus-like particles . Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used . Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate . The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles . The virus particles are 80-130 nm in size . The feed streams were flocculated using a cationic polyelectrolyte . Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes . The level of virus clearance in the permeate was determined . For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells . While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams . Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant . However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes . The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration . Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles . Flocculation of the feed stream leads to an increase in the permeate flux . 2004 Wiley Periodicals, Inc.

Yan Ke Xue Bao, 2002 Jun, 18(2), 110 - 4
{A pilot study of bone marrow stromal cells intraocular transplantation in the S334 transgenic rats and Sprague-Dawley rats}; Qiu G et al.; PURPOSE: To investigate the survival and differentiation of human bone marrow stromal cells (BMSC) intraocular transplantation in newborn S334 retinal degeneration transgenic rats and (Sprague-Dawley)SD rats . METHODS: Human bone marrow stromal cells line was grown on the adhesive substrate in the condition media including a-Modified Eagle medium (a-MEM)/10% fetal bovine serum . The experiments were divided into four groups: Group 1: BMSC + (Retinoid Acid) RA transplanted in S334 transgenic rats (n = 5); Group 2: BMSC transplanted in S334 transgenic rats (n = 5); Group 3: BMSC + RA transplanted in SD rats (n = 5); Group 4: BMSC transplanted in SD rats (n = 5) . 2 microl cell suspension (about 4 x 10(4) cells) was injected into the vitreous space in the transgenic rats and normal SD rats at Postnatal 1 (P1) respectively . The right eyes were treated eyes and the left eyes were used as control . At P14 and P23, the rats were killed and enucleated for histological assays using plastic section . RESULTS: In Group 1, the transplanted cells were well survived . They could continue to differentiate and participate in late-stage retinal development . The number of inner nuclear layer increased . Moreover, the host retina increased their thickness, but photoreceptor cells were not rescued from transplant . In Group 2, at P14, the BMSC continue to differentiate toward their linage cell fate and formed into hemorrhage island structures with few neurons if RA was not applied . Group 3, BMSC could survive, migrate . The number of inner nuclear layer increased also . In Group 4, it revealed that host retina structures were disorganized and transplant cells formed atypical proliferating mass . CONCLUSIONS: This pilot experiment indicated that bone marrow stromal cells could survival, differentiate and participate in the retinal development after transplanted into vitreous space in the new born transgenic rats and SD rats . Histological assays showed that transplanted cells integrated with inner nuclear layer of host retina . Thus, bone marrow stromal cells may be a useful vehicle for auto- transplantation for the therapy of variety of retinal degenerative disorders.

Plant Cell, 2004 Nov, 16(11), 3020 - 32 Epub 2004 Oct 19.
Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis; Menard R et al.; Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals . We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana . In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst . Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems . In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation . Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis . Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins . In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent . Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active . In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance . The results open new routes to work out new molecules suitable for crop protection.

Methods Mol Med, 2004, 107, 147 - 62
Purification of peripheral blood natural killer cells; Perussia B et al.; The ability to perform biological studies on natural killer (NK) cells requires effective methods for their isolation from hematopoietic cells of other lineages . NK cells are a discrete lymphocyte subset distinct from B and T cells based on both physical and phenotypic characteristics that can be exploited for their purification . Techniques based on differential cell buoyancy {centrifugation on discontinuous density gradients such as Percoll } have been used to enrich NK cells from mixed lymphocyte populations but do not allow their purification to homogeneity . The mononuclear cell suspensions obtained, although enriched in NK cells, also contain variable proportions of other cell types (monocytes and/or activated T and B cells) , and subsets of NK cells of higher density are lost in these preparations.The most satisfactory purification techniques for NK cells, as well as for other leukocyte subsets, rely on their distinctive phenotype and make use of monoclonal antibodies (MAbs) directed to lineage-specific surface antigens (Ag) . NK cell-specific surface markers have not been identified yet . Lack of surface expression of T-cell receptor/CD3 complex and surface immunoglobulins (Ig), concomitant with expression of CD16 (low-affinity receptor for the Fc portion of IgG, FcgammaRIIIA) , CD56 (an N-CAM isoform) , and CD161 (NKRP-1A) (expressed, unlike CD56 and CD16, also on NK cells at a relatively developmentally immature functional stage) identify NK cells within mononuclear cell populations . MAbs to these three differentiation Ag are available, and cells sensitized with them can be detected with a variety of secondary reagents to permit their identification and physical separation . Using MAbs, homogeneous NK cell preparations are isolated from mixed mononuclear cell populations following either of two schemes: (1) direct isolation using MAbs to surface Ag expressed on these cells (positive selection), or (2) depletion of all cells other than NK using a mixture of MAbs directed to Ag expressed on the former but absent from the latter population (negative selection) . The advantage of positive selection is the rapid and specific isolation of NK cells . However, Ab binding to antigens capable of signal transduction, e.g., CD16, may lead to modulation of NK cells' biological functions, making them unusable for selected applications.

Methods Mol Med, 2004, 107, 137 - 46
Human mononuclear phagocytes in tissue culture; Keisari Y; Peripheral blood human monocytes (HuMo) are the major source for human mononuclear phagocytes . Such monocytes when cultured, differentiate into monocyte-derived macrophages (HuMoDM), and undergo various structural, biochemical, and functional changes.The most common method used for the separation of mononuclear cells from the blood, is the Ficoll-Hypaque density gradient centrifugation, essentially described by Boyum . Ficoll-Hypaque at a density of 1.077 g/L is used to separate the denser granulocytes and erythrocytes from the lighter lymphocytes, monocytes, and thrombocytes . The mononuclear cells stay at the top of the Ficoll-Hypaque layer, whereas the denser cells sink to the bottom of the centrifuge tube.Peripheral blood monocytes are purified from the mononuclear fraction by adherence to plastic . Adherence can be carried out either directly onto tissue-culture plates in which they will be further grown (24- or 96-well plates), or onto tissue-culture flasks from which they will be hence removed and recultured in the required plates or chambers.The adherent human MoDM bind firmly to plastic substrata and it is very difficult to remove the cells for quantitative measurements . There are several methods to enumerate or quantitate adherent monocytes/macrophages of which four are described below.When an enriched monocyte cell suspension is required, MNC harvested on Ficoll-Hypaque gradients can be further separated on Percoll gradients into lymphocytes and monocytes . The method initially described by Ulmer and Flad , was modified by Orlandi et al . , which used only one Percoll concentration . After separation on Percoll, the monocytes that are less dense than lymphocytes stay on top of the Percoll layer, whereas the lymphocytes go through to the bottom of the tube.Long-term incubation (7-21 d) of monocytes under tissue-culture conditions results in the differentiation of the cultured cells, and in the appearance of HuMoDM . Yet, long-term incubation of the cells in culture results in a substantial loss of cells . In various studies, it was found that cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 , or IL-4, as well as PKC activators/tumor promoters facilitate the survival and differentiation of human monocytes.

Methods Mol Med, 2004, 107, 13 - 28
Cultivation of normal human epidermal melanocytes in the absence of phorbol esters; Hsu MY et al.; An important approach in studies of normal, diseased, and malignant cells is their growth in culture . The isolation and subsequent culture of human epidermal melanocytes has been attempted since 1957 , but only since 1982 have pure normal human melanocyte cultures been reproducibly established to yield cells in sufficient quantity for biological, biochemical, and molecular analyses . Selective growth of melanocytes, which comprise only 3-7% of epidermal cells in normal human skin, was initially achieved by suppressing the growth of keratinocytes and fibroblasts in epidermal cell suspensions with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the intracellular cyclic adenosine 3', 5' monophosphate (cAMP) enhancer cholera toxin, respectively, which both also act as melanocyte growth promoters . However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses . Recent progress in basic cell-culture technology, along with an improved understanding of culture requirements, has led to an effective and standardized isolation method, and special TPA-free culture media for selective growth and long-term maintenance of human melanocytes . The detailed description of this new method is aimed at encouraging its use in basic and applied biological research.

Arch Dermatol, 2004 Oct, 140(10), 1211 - 5
Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte-keratinocyte cell transplantation; Mulekar SV; OBJECTIVE: To evaluate long-term efficacy and safety of melanocyte-keratinocyte cell transplantation in the management of segmental and focal vitiligo . DESIGN: A simpler and modified method based on that of Olsson and Juhlin was performed . This method uses a shaved biopsy skin sample up to one tenth the size of the recipient area . The skin sample is incubated, and the cells are mechanically separated using trypsin-EDTA solution and then centrifuged to prepare a suspension . Cell suspension is then applied to the dermabraded depigmented skin area, and a collagen dressing is applied to keep it in place . PATIENTS: Fifty patients with segmental and 17 with focal vitiligo were treated . One patient with segmental and 2 with focal vitiligo did not attend any follow-up visits . The remaining patients were observed for a period of up to 5 years . INTERVENTION: Autologous, noncultured melanocyte-keratinocyte cell transplantation . MAIN OUTCOME MEASURE: Repigmentation was graded as excellent with 95% to 100% pigmentation, good with 65% to 94%, fair with 25% to 64%, and poor with 0% to 24% of the treated area . RESULTS: In the segmental vitiligo group, 41 patients (84%) showed excellent, 3 (6%) good, and 5 (10%) poor pigmentation, which was retained until the end of the respective follow-up period . In the focal vitiligo group, 11 patients (73%) showed excellent, 1 (7%) fair, and 3 (20%) poor pigmentation, which was retained until the end of the respective follow-up period . CONCLUSIONS: Melanocyte-keratinocyte cell transplantation is a simple, safe, and effective surgical therapy . Patients with segmental and focal vitiligo can experience a prolonged disease-free period, which may extend through the rest of their lives.

Arch Dermatol, 2004 Oct, 140(10), 1203 - 8
Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo; van Geel N et al.; OBJECTIVES: To investigate the efficacy of epidermal noncultured cellular grafting in patients with vitiligo and the role of postinflammatory, spontaneous, or UV-induced pigmentation in obtaining repigmentation . DESIGN: A prospective, randomized, double-blind, placebo-controlled study . SETTING: Ambulatory patients in an institutional practice . Patients were followed up for 3 to 12 months . PATIENTS: A total of 33 paired, symmetrically distributed leukodermic lesions, all resistant to therapy, were observed in 28 patients . Nineteen patients appeared to have a stable vitiligo (group 1), whereas there was doubt about the stability of the disease in 9 patients (group 2) . INTERVENTION: After laser ablation, a hyaluronic acid-enriched cellular graft was applied to 1 lesion while the paired lesion received placebo . Three weeks later all lesions were exposed to UV irradiation twice per week for approximately 2 months . MAIN OUTCOME MEASURES: Primarily, the percentage of repigmentation was assessed after 3, 6, and 12 months using a digital image analysis system . The repigmentation pattern was also evaluated after 1 and 3 months . RESULTS: A strongly significant difference between cellular grafts and placebo was observed after 3, 6, and 12 months (P<.001, P = .002, and P = .002, respectively) . In group 1, repigmentation of at least 70% of the treated area was achieved in 55%, 57%, and 77% of the actively treated lesions 3, 6, and 12 months after treatment, whereas in group 2 repigmentation of at least 70% of the treated area was not observed at any time point . The repigmentation pattern was diffuse in 94% of the responding patients . CONCLUSIONS: After a strict preoperative selection for disease stability, transplantation resulted in repigmentation of at least 70% of the treated area in most actively treated vitiligo lesions . Repigmentation was primarily caused by the transplanted melanocytes.

Anal Chem, 2004 Oct 15, 76(20), 6137 - 43
Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device; Irimia D et al.; Investigating the intracellular contents of single cells is essential for understanding physiologic and pathologic processes at the cellular level . While existing protocols for cell lysis and sample preparation work well for larger samples, scaling to a single-cell level is challenging because of unavoidable analyte dilution and losses . Thus, we are proposing a microfabricated device for the controlled handling and mixing of picoliter cell suspension and lysis solution volumes . Cells and fluids are independently isolated in two microchambers of 25-pL volumes using the geometry of the microchannels and the coordinated action of four on-chip thermopneumatic actuators . Virtual walls formed by liquid-air interfaces in the hydrophobic capillary separate the two volumes, which are subsequently allowed to mix after drawing the air out of the capillary connecting the two microchambers . Following cell lysis, a limited and stable dilution of intracellular components is achieved, simplifying the requirements for subsequent analysis . Two assays at single-cell level, one for direct estimation of the intracellular concentration of a soluble dye and the other for indirect evaluation of intracellular quantities of insoluble actin, demonstrate the use of the microfabricated device for single-cell assays.

Clin Lab, 2004, 50(9-10), 575 - 80
Use of the enzyme method for antibody identification; Strobel E; Enzyme methods for red blood cell antibody testing may have two goals: detection of weak antibodies by increasing the strength of the reactions and differentiation of the antibodies in an antibody mixture by abolishing the reaction of antibodies against enzyme-labile antigens . We analyzed the phenotype listing sheets of all lots of one year (expiration date in 2002) of 8 products (5 manufacturers) (together 130 worksheets) . The aim was to find out how often some antibodies could only be detected after enzyme treatment, when there is an additional antibody against one of the following enzyme-labile antigens in the patient's serum: Fya, Fyb, M, N, S and s . If there is one of these antibodies against an enzyme-labile antigen, an additional antibody against one of the following enzyme-stable antigens D, C, E, c, e, CW, K, Kpa, Jsa, Jka, Jkb, Lea, Leb, P1, Lua cannot be detected on average in 20% of the panels . Moreover, in a further 37% of the panels there is no red blood cell suspension carrying the Kpa, in 44% none carrying the Jsa and in 19% no one carrying the Lua antigen . On the other hand, an antibody against one of the high-incidence antigens k, Kpb, Jsb or Lub can be detected in each of the 130 panels, also if there is an additional antibody directed against one of the above-mentioned enzyme-labile antigens . As also nowadays in some antibody identification panels antibodies against some enzyme-stable antigens might be covered by antibodies against enzyme-labile antigens, the enzyme method can be a helpful completion in antibody differentiation in some sera with multiple antibodies.

Eur Biophys J . 2004 Oct 5; {Epub ahead of print}
Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles; Liu Y et al.; We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles gamma of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm(2) on the surface of a horn tip . Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t' from 0 to 60 s . HeLa-S3 viability decreased as a single exponential function of the total exposure time t= gammat' with a common time constant tau=3.8 s for three duty cycles . Transfection was evaluated by counting the number of beta-galactosidase(beta-Gal)-positive cells relative to the total number of cells . Pulsed ultrasound provided an enhanced transfer of the beta-Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control . The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t= tau with gamma=10, 20 and 30%, respectively . The number ratio of beta-Gal-positive cells to the surviving cells after exposure increased with t' according to a modified logistic equation . The degree of hemolysis also increased exponentially with t' at a time constant tau'= tau(0)/ gamma for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with tau(0)=0.9 s . Thus the total exposure time for the optimal transfection efficiency was tau, that is, nearly four times of tau(0) . Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.

Biol Trace Elem Res, 2004 Sep, 100(3), 229 - 45
Analyses of impact of metal ion contamination on carp (Cyprinus carpio L.) gill cell suspensions; Arabi M; The decline in fish population because of water contamination is problem . As a result of direct exposure in water, it has been readily accepted that the gills are the main site of water contamination and toxicity (e.g., metal ions) . In the present study, we investigated metal ion contamination on the functional capacity of carp gill cells with antioxidant interactions in an in vitro study . The extent of cellular membrane damage, lipid peroxidation (LPO) (as TBARS levels), and glutathione (GSH) content were investigated after the addition of two metal ion compounds (viz., CuSO(4) and HgCl(2)) in various concentrations (300, 500, 700, 1000, and 3000 microM) to gill cell preparation of the freshwater fish carp (Cyprinus carpio L.) with modulations by bovine serum albumin (BSA) (0.5% and 1.0%) and dimethyl sulfoxide (DMSO) (0.5%) as free-radical scavengers . The Comet assay technique was also performed for the highest concentrations of the two mentioned metal ions as an index of DNA breaks . The outcomes were as follows: (1) Copper and mercury increased the rate of LPO dose dependently (r=+0.995 and r=+0.993, respectively; p<0.001), but the GSH content was only marginally affected (r = -0.787 and r = -0.844, respectively; p <0.05) . (2) Depletion of GSH molecules by copper had a wider range than mercury . (3) In the highest concentration of metal ions (3000 microM), both DMSO and 1.0% BSA showed a pro-oxidative potential to elevate the levels of TBARS (p<0.001), but for other concentrations when supplemented with three scavengers, a fall in the levels of the latter was found . (4) The addition of 1.0% BSA to medium containing 3000 microM of metal ions caused a significant decline in GSH content (p<0.01) . (5) Copper and mercury could cause a high rate of DNA breaks (single stranded) in carp gill cell suspensions as a Comet appearance.These findings indicate that copper and mercury have a deleterious influence on membrane integrity and GSH content in a relatively dose-dependent manner . The complexes of metal ions and thiol (SH) residues of cell proteins could also act as a potential cell toxicant leading to disturbances in cell functions causing cell death . DNA fragmentation is frequent in metal ion contamination.

Biomaterials, 2005 Apr, 26(11), 1287 - 92
Poly(N-isopropylacrylamide)-graft-polypropylene membranes containing adsorbed antibody for cell separation; Okamura A et al.; We developed a novel selective cell-separation method based on using a poly(N-isopropylacrylamide)-graft-polypropylene (PNIPAAm-g-PP) membrane containing adsorbed monoclonal antibody specific to the target cell . This membrane was prepared by plasma-induced polymerization and soaking in an antibody solution at 37 degrees C . Poly(N-isopropylacrylamide) has a thermoresponsive phase transition: at 32 degrees C water-insoluble (hydrophobic) and water-soluble (hydrophilic) states interconvert . Adsorption of antibody onto PNIPAAm-g-PP membrane at 37 degrees C and its desorption at 4 degrees C was verified by fluorescence-microscopy of the PNIPAAm-g-PP membrane after soaking it in fluorescein-conjugated goat anti-mouse IgG in phosphate-buffered saline . PNIPAAm-g-PP membranes containing adsorbed anti-mouse CD80 monoclonal antibody preferentially captured mouse-CD80 transfected cells at 37 degrees C compared with membranes lacking antibody or containing anti-mouse CD86 monoclonal antibody . Detachment of captured cells from PNIPAAm-g-PP membranes was facilitated by washing at 4 degrees C because of the thermoresponsive phase transition of PNIPAAm . With this method, mouse CD80- or mouse CD86-transfected cells were enriched from a 1:1 cell suspension to 72% or 66%, simply and with high yield.

FEBS Lett, 2004 Oct 8, 576(1-2), 266 - 70
Heterologously expressed protein phosphatase calcineurin downregulates plant plasma membrane H+-ATPase activity at the post-translational level; Marin-Manzano MC et al.; To investigate the effects of calcineurin expression on cellular ion homeostasis in plants, we have obtained a transgenic cell culture of tomato, expressing constitutively activated yeast calcineurin . Transgenic cells exhibited reduced growth rates and proton extrusion activity in vivo . We show that reduction of plasma membrane H+-ATPase activity by expression of calcineurin is the basis for the observed phenotypes . Transgenic calli and cell suspensions displayed also increased salt tolerance and contained slightly higher Ca2+ and K+ levels . This demonstrates that calcineurin can modulate ion homeostasis in plants as it does in yeast by affecting the activity of primary ion transporters .

J Environ Sci Health B, 2004 May, 39(4), 533 - 49
Metabolism of the nonylphenol isomer {ring-U-14C}-4-(3',5'-dimethyl-3'-heptyl)-phenol by cell suspension cultures of Agrostemma githago and soybean; Schmidt B et al.; The biotransformation of the nonylphenol isomer {ring-U-14C}-4-(3',5'-dimethyl-3'-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures . With the A . githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase . Initial concentrations of {14C}-4-353-NP were 1 mg L(-1) (soybean), and 5 and 10 mg L(-1) (A . githago) . After 2 (soybean) and 7 days (A . githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media . The latter two products emerged especially in soybean cultures . Portions of primary metabolites amounted to 19-22% (soybean) and 21-42% of applied 14C (A . githago) . After liberation from their glycosides, the primary 4-353-NP metabolites formed by A . githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives . In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP . Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative . Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible . The main primary metabolites produced by A . githago were supposed to be four diastereomers of 6'-hydroxy-4-353-NP (about 80% of all products identified) . It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.

J Biol Chem, 2004 Dec 17, 279(51), 52940 - 8 Epub 2004 Oct 01.
Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus; Pauw B et al.; In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract . To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter . This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C . roseus, ZCT1, ZCT2, and ZCT3 . These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays . In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter . The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate . These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C . roseus.

Int J Radiat Oncol Biol Phys, 2004 Nov 1, 60(3), 920 - 7
Combination of the vascular targeting agent ZD6126 with boron neutron capture therapy; Masunaga S et al.; PURPOSE: The aim of this study was to evaluate the antitumor efficacy of the vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) in the rodent squamous cell carcinoma (SCC) VII carcinoma model, in combination with boron neutron capture therapy (BNCT) . METHODS AND MATERIALS: Sodium borocaptate-(10)B (BSH, 125 mg/kg, i.p.) or l-p-boronophenylalanine-(10)B (BPA, 250 mg/kg, i.p.) was injected into SCC VII tumor-bearing mice, and 15 min later, ZD6126 (100 mg/kg, i.p.) was administered . Then, the (10)B concentrations in tumors and normal tissues were measured by prompt gamma-ray spectrometry . On the other hand, for the thermal neutron beam exposure experiment, SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, followed by treatment with a (10)B-carrier and ZD6126 in the same manner as the above-mentioned (10)B pharmacokinetics analyses . To obtain almost similar intratumor (10)B concentrations during neutron exposure, thermal neutron beam irradiation was started from the time point of 30 min after injection of BSH only, 90 min after BSH injection for combination with ZD6126, 120 min after the injection of BPA only, and 180 min after BPA injection for combination with ZD6126 . Right after irradiation, the tumors were excised, minced, and trypsinized . The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent {Q} cells) was determined using immunofluorescence staining for BrdU . Meanwhile, the MN frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU . The clonogenic cell survival assay was also performed in mice given no BrdU . RESULTS: Pharmacokinetics analyses showed that combination with ZD6126 greatly increased the (10)B concentrations in tumors after 60 min after BSH injection and after 120 min after BPA injection . The concentrations of (10)B from BSH in normal tissues were also raised by combination with ZD6126, although not so clearly as those in tumors . Combination with ZD6126 had almost no effect on the concentrations of (10)B from BPA in normal tissues . The clonogenic surviving fractions of total tumor cells and the MN frequencies of both total and Q tumor cells were reduced and increased by combination with ZD6126, respectively, whether BSH or BPA was employed . However, the degrees of these changes in the clonogenic surviving fractions and the MN frequencies were more obviously observed in tumors from BSH-injected mice than from BPA-injected mice, and in Q tumor cells than in total tumor cells regardless of the employed (10)B-carrier . CONCLUSIONS: Combination with ZD6126 was regarded as more promising in BSH-BNCT than BPA-BNCT, and more effective for enhancing the sensitivity of the Q tumor cells than that of the total tumor cells . This resulted in the decrease in the extended difference in the sensitivity between the total and Q tumor cells caused by the use of (10)B-carrier for BNCT.

Wei Sheng Yan Jiu, 2004 Jul, 33(4), 494 - 6
{Comparison between effects of mechanical mince on the DNA damages of spleen, liver and kidney cells in mice}; Li X et al.; OBJECTIVE: To study whether there are differences between effects of the DNA damages of the spleen, liver and kidney cells in mice caused by mechanical mince and the spleen, liver and kidney in mice can be dissociated into single cells by mechanical mince or not . METHODS: No drug was administrated to the tested group mice, and Cyclophosphamide was administrated to the control group mice with 150 mg/kg doses . Then the three kinds of single cell suspensions were prepared by mechanical mince . The cell livabilities were detected by trypanblau coloration, and effects of mechanical mice on the DNA damages of the three kinds of cells were detected by single cell gel electrophoresis . RESULTS: The single cell quantity prepared from spleen, liver and kidney in short time by mechanical mince can meet completely the needs of comet assay . The percentages of cells with comets of the tested group were respectively 3.20%, 6.21%, 9.22% and the DNA migrations were respectively 27.30microm, 28.45microm, 47.10microm . There were significant differences between the percentages of cells with comets in the tested groups (P < 0.05) . In the DNA migration test, there were significant differences between the spleen cells and kidney cells or between liver cells and kidney cells of the three tested groups (P < 0.05) . The percentages of cells with comets and the DNA migrations of the three types cells in tested groups were low significantly (P < 0.01, P < 0.001) compared with the corresponding positive control groups . CONCLUSION: The effects of mechanical mince on the DNA damages of spleen, liver and kidney cells became stronger and stronger orderly . Spleen and liver can be dissociated into single cells by mechanical mince more appropriately.

Endocrinology, 2005 Jan, 146(1), 71 - 6 Epub 2004 Sep 30.
Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro; Rotllant J et al.; The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system . Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner . The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH . In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production . The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately . The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and {H3}myo-inositol incorporation in an interrenal cell suspension . Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways . These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.

Vestn Ross Akad Med Nauk, 2004, (8), 15 - 8
{Pulmonary cell susceptibility in mice and rats to influenza virus when infected in vivo and in vitro}; Sergeev AA et al.; The purpose of the case study was to evaluate comparatively the relative contribution of cell susceptibility and the inhibiting effect of factors of pulmonary epithelial lining in mice and rats to influenza virus A/Aichi/2/68 (H3N2) adapted to mice as related with the development of infection process in the lungs of experimental animals when infected in vivo and in vitro . Mice and rats were infected aerogenically with different doses of influenza virus . The primary cell-culture suspensions sampled from the lungs of mice and rats were used to study the adsorption and dynamics of influenza virus production in infection by different dose of influenza virus in vitro . The cell suspensions were shown to be able to produce the influenza virus for as long as 48 hours after infection . It was for the first time that the results denoted the identical susceptibility of primary pulmonary cells in mice and rats to influenza virus . A lower pulmonary susceptibility to influenza virus in rats versus mice could be indicative of that the surface factors of epithelial lining contribute essentially to shaping the pulmonary susceptibility to influenza virus since there is no difference of the susceptibility of pulmonary cells to influenza virus between the two above animals' species.

Plant Cell Rep, 2005 Jan, 23(8), 573 - 8 Epub 2004 Sep 29.
Effects of boron deficiency in cell suspension cultures of Populus alba L; Kakegawa K et al.; Cell suspension cultures of Populus alba L . (original cells) require at least 10 muM boron for appropriate growth . Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 muM) . The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 muM boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former . The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid . Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.

Biophys J, 2004 Oct, 87(4), 2322 - 34
Rheology and ultrasound scattering from aggregated red cell suspensions in shear flow; Haider L et al.; The shear flow dynamics of reversible red cell aggregates in dense suspensions were investigated by ultrasound scattering, to study the shear disruption processes of Rayleigh clusters and examine the effective mean field approximation used in microrheological models . In a first section, a rheo-acoustical model, in the Rayleigh scattering regime, is proposed to describe the shear stress dependence of the low frequency scattered power in relation to structural parameters . The fractal scattering regime characterizing the anisotropic scattering from flocs of size larger than the ultrasound wavelength is further discussed . In the second section, we report flow-dependent changes in the low-frequency scattering coefficient in a plane-plane flow geometry to analyze the shear disruption processes of hardened or deformable red cell aggregates in neutral dextran polymer solution . Rheo-acoustical experiments are examined on the basis of the rheo-acoustical model and the effective medium approximation . The ability of ultrasound scattering technique to determine the critical disaggregation shear stress and to give quantitative information on particle surface adhesive energy is analyzed . Lastly, the shear-thinning behavior of weakly aggregated hardened or deformable red cells is described .

Plant J, 2004 Oct, 40(2), 302 - 13
Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis; Samanani N et al.; (S)-Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine, sanguinarine, and berberine, in plants . A molecular clone encoding NCS was isolated from a meadow rue (Thalictrum flavum ssp . glaucum) cell suspension culture cDNA library . Heterologous expression of the NCS cDNA, truncated to remove a putative signal peptide, produced a recombinant protein with NCS activity . Recombinant NCS showed sigmoidal saturation kinetics for dopamine (Hill coefficient=1.98), hyperbolic saturation kinetics for 4-HPAA (Km of 700 microm), and pH and temperature optima of 7.0 and 40 degrees C, respectively, all similar to the purified, plant-derived enzyme . NCS exhibits 28-38% identity, and putative structural homology, with the Bet v 1 allergen and pathogenesis-related (PR)10 protein families . NCS also displays 35% identity with the enzyme (HYP1) responsible for hypericin biosynthesis in St John's wort (Hypericum perforatum) . The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes within the Bet v 1 and PR10 protein families . Weaker homology was also detected between NCS and proteins identified in the latex of Papaver somniferum (opium poppy), and in Arabidopsis thaliana . A family of three to five NCS genes is abundantly expressed in the rhizome, followed by petioles and roots of T . flavum . NCS transcripts were localized to the immature endodermis and pericycle in roots, and the protoderm of leaf primordia in rhizomes; thus, the sites of NCS gene expression and berberine accumulation are temporally and spatially separated in roots and rhizomes respectively.

Immunohematol, 1995 Sep, 11(3), 71 - 3
ABO discrepancy with monoclonal ABO reagents caused by a pH-dependent autoantibody; Kennedy MS et al.; ABO discrepancy was noted when a patient's unwashed saline red cell suspension was tested with monoclonal ABO reagents . The discrepancy was resolved when a washed (X 3) saline red cell suspension was used in repeat testing . The patient's serum contained an autoagglutinin that reacted optimally below pH 7.0 . The discrepancy occurred when monoclonal ABO reagents, formulated at a low pH, lowered the pH of the reaction mixture, and autoagglutination was observed . Immunohematology 1995;11:71-73.

J Biomed Opt, 2004 Sep-Oct, 9(5), 982 - 94
Measuring red blood cell flow dynamics in a glass capillary using Doppler optical coherence tomography and Doppler amplitude optical coherence tomography; Moger J et al.; Blood, being a suspension of deformable red cells suspended in plasma, displays flow dynamics considerably more complicated than those of an ideal Newtonian fluid . Flow dynamics in blood capillaries of a few hundred micrometers in diameter are investigated using Doppler optical coherence tomography (DOCT) and Doppler amplitude optical coherence tomography (DAOCT), a novel extension of DOCT . Velocity profiles and concentration distributions of normal and rigidified in vitro red blood cell suspensions are shown to vary as functions of mean flow velocity, cell concentration, and cell rigidity . Deviation from the parabolic velocity profile expected for Pouseille flow is observed for both rigid and normal cells at low flow rates . Axial red cell migration both toward and away from the tube axis is observed for both rigid and normal cells as a function of flow velocity . Good agreement is found between our measurements, and theoretical expectations . (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Gene Ther, 2005 Jan, 12(1), 67 - 74
Shuttle of lentiviral vectors via transplanted cells in vivo; Blomer U et al.; Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo . Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy . A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation . Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells . We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells . To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle . We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions . Those lentiviral vector particles were able to transduce target cells in transwell experiments . Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo . Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.

IEEE Trans Nanobioscience, 2004 Jun, 3(2), 90 - 5
A parallel-plate flow chamber to study initial cell adhesion on a nanofeatured surface; Martines E et al.; Cells in the human body come across many types of information, which they respond to . Both material chemistry and topography of the surface where they adhere have an effect on cell shape, proliferation, migration, and gene expression . It is possible to create surfaces with topography at the nanometric scale to allow observation of cell-topography interactions . Previous work has shown that 100-nm-diameter pits on a 300-nm pitch can have a marked effect in reducing the adhesion of rat fibroblasts in static cultures . In the present study, a flow of cell suspension was used to investigate cell adhesion onto nanopits in dynamic conditions, by means of a parallel-plate flow chamber . A flow chamber with inner nanotopography has been designed, which allows real-time observation of the flow over the nanopits . A nanopitted pattern was successfully embossed into polymethylmethacrylate to meet the required shape of the chamber . Dynamic cell adhesion after 1 h has been quantified and compared on flat and nanopitted polymethylmethacrylate substrates . The nanopits were seen to be significantly less adhesive than the flat substrates (p < 0.001), which is coherent with previous observations of static cultures.

Phytochemistry, 2004 Sep, 65(17), 2455 - 61
Stimulating the production of homoisoflavonoids in cell suspension cultures of Caesalpinia pulcherrima using cork tissue; Zhao P et al.; It has previously been demonstrated that cork tissue increases the efficiency of the production of lipophilic secondary metabolites in diverse plant cell suspension cultures . In the present study, three new homoisoflavonoids--named dihydrobonducellin, 2'-methoxydihydrobonducellin, and 2'-methoxybonducellin--and bonducellin and isobonducellin were isolated from Caesalpinia pulcherrima cultured cells coincubated with cork tissue . Cork tissue increased the production of 2'-methoxybonducellin by about 7-fold relative to control cells, and more than 80% of the product was recoverable from the cork tissue . When cork tissue and methyl jasmonate or yeast extract were added simultaneously to the medium, the amount of 2'-methoxybonducellin produced increased further . The production of the other four homoisoflavonoids was enhanced by variable amounts . Our results indicate that the addition of cork tissue would be an effective technique for investigating formation of secondary metabolites that usually accumulate only in trace amounts .

Int J Radiat Oncol Biol Phys, 2004 Oct 1, 60(2), 570 - 7
Effects of mild temperature hyperthermia and p53 status on the size of hypoxic fractions in solid tumors, with reference to the effect in intratumor quiescent cell populations; Masunaga S et al.; PURPOSE: To determine the effects of mild temperature hyperthermia (MTH) and p53 status of tumor cells on the size of hypoxic fractions (HFs) in solid tumors, with reference to the effect on intratumor quiescent (Q) cell populations . METHODS AND MATERIALS: Human head-and-neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into left hind legs of Balb/cA nude mice . Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors . The mice then received nicotinamide injection or carbogen gas (95% O(2), 5% CO(2)) inhalation combined with or without MTH . Nicotinamide prevents intermittent blood flow that could induce perfusion-limited acute hypoxia . Chronically hypoxic cells in regions beyond the limitation of oxygen diffusion in tumors are oxygenated by increasing the oxygen transport capacity of circulating blood with carbogen gas inhalation . After each treatment, the mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain HFs in the tumors . Immediately after irradiation, the tumors were excised, minced, and trypsinized . The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B) to inhibit cytoplasmic division while allowing nuclear division . Tumor cells not labeled with BrdU were detected with immunofluorescence staining of BrdU for P cells, and the micronucleus frequency in cells without BrdU labeling { = Q cells} was determined . The micronucleus frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU . RESULTS: SAS/mp53 tumors showed larger values for the size of not only the HF but also the diffusion-limited chronically HF than SAS/neo tumors . Q cell populations included a larger HF, particularly the chronically HF, than total cell populations in both tumors, especially in SAS/neo tumors . MTH could efficiently oxygenate the chronically HF, irrespective of p53 status . CONCLUSION: MTH is a useful combined treatment with a radioenhancement effect on intratumor Q cells, irrespective of the p53 status of tumor cells . The p53 status has the potential to affect microenvironmental conditions within solid tumors.

Eur J Neurosci, 2004 Oct, 20(7), 1695 - 704
Adult neural progenitor cells provide a permissive guiding substrate for corticospinal axon growth following spinal cord injury; Pfeifer K et al.; Adult neural progenitor cells (NPC) are an attractive source for cell transplantation and neural tissue replacement after central nervous system (CNS) injury . Following transplantation of NPC cell suspensions into the acutely injured rat spinal cord, NPC survive; however, they migrate away from the lesion site and are unable to replace the injury-induced lesion cavity . In the present study we examined (i) whether NPC can be retained within the lesion site after co-transplantation with primary fibroblasts, and (ii) whether NPC promote axonal regeneration following spinal cord injury . Co-cultivation of NPC with fibroblasts demonstrated that NPC adhere to fibroblasts and the extracellular matrix produced by fibroblasts . In the presence of fibroblasts, the differentiation pattern of co-cultivated NPC was shifted towards glial differentiation . Three weeks after transplantation of adult spinal-cord-derived NPC with primary fibroblasts as mixed cell suspensions into the acutely injured cervical spinal cord in adult rats, the lesion cavity was completely replaced . NPC survived throughout the graft and differentiated exclusively into glial cells . Quantification of neurofilament-labeled axons and anterogradely labeled corticospinal axons indicated that NPC co-grafted with fibroblasts significantly enhanced axonal regeneration . Both neurofilament-labeled axons and corticospinal axons aligned longitudinally along GFAP-expressing NPC-derived cells, which displayed a bipolar morphology reminiscent of immature astroglia . Thus, grafted astroglial differentiated NPC promote axon regrowth following spinal cord injury by means of cellular guidance.

Cryo Letters, 2004 Jul-Aug, 25(4), 265 - 72
Improved methods for controlled rapid cooling of cell suspensions; Morris GJ et al.; Simple, reproducible, methods of achieving rapid rates of cooling in the range 100 degrees C min(-1) to 1000 degrees C min(-1) are described for straws and cryovial . These methods use the direct contact of straws or cryovials with pre-cooled granules or plates as the heat sink . Liquid nitrogen may be adsorbed into suitable material, for example activated charcoal, zeolites and molecular sieves as the matrix to achieve rapid cooling . Controllable rapid rates of cooling may also be attained by using non-adsorbants . The rate of cooling may be modified by changing the adsorbant material, the size of the adsorbant granules and the temperature of the adsorbant.

Immunohematol, 2001 Jun, 17(2), 53 - 6
Comparison of three low-ionic diluents for dilution and storage of reagent A1 and B cells for testing in gel technology; Steiner EA et al.; Currently, ABO serum grouping performed by gel technology employs a red cell diluent containing EDTA (MTS Diluent 2 Plus trade mark ) that does not permit extended storage of the red cell suspensions . A diluent currently used for suspension and long-term storage of reagent red cells for antibody detection and identification (Ortho 0.8% Red Cell Diluent trade mark ) was evaluated for use with A1 and B cells . Because this diluent does not contain EDTA, testing was limited to EDTA samples . As a comparison, a Micro Typing Systems (MTS) diluent not containing EDTA (MTS Diluent 2 trade mark ) was also tested . MTS-suspended red cells were maintained for 24 hours and compared with Ortho-Clinical Diagnostics 0.8% suspended red cells maintained for 7 days . ABO serum grouping was performed on 144 EDTA plasma samples using all three cell suspensions . Acceptable results were noted in all aspects of testing . Immunohematology 2001;17: 53-56.

Scand J Clin Lab Invest, 2004, 64(6), 579 - 87
LEGO bricks used as chemotactic chambers: evaluation by a computer-assisted image analysis technique; Azzara A et al.; One of the main techniques used to explore neutrophil motility, employs micropore filters in chemotactic chambers . Many new models have been proposed, in order to perform multiple microassays in a rapid, inexpensive and reproducible way . In this work, LEGO bricks have been used as chemotactic chambers in the evaluation of neutrophil random motility and chemotaxis and compared with conventional Boyden chambers in a "time-response" experiment . Neutrophil motility throughout the filters was evaluated by means of an image-processing workstation, in which a dedicated algorithm recognizes and counts the cells in several fields and focal planes throughout the whole filter; correlates counts and depth values; performs a statistical analysis of data; calculates the true value of neutrophil migration; determines the distribution of cells; and displays the migration pattern . By this method, we found that the distances travelled by the cells in conventional chambers and in LEGO bricks were perfectly identical, both in random migration and under chemotactic conditions . Moreover, no interference with the physiological behaviour of neutrophils was detectable . In fact, the kinetics of migration was identical both in random migration (characterized by a gaussian pattern) and in chemotaxis (characterized by a typical stimulation peak, previously identified by our workstation) . In conclusion, LEGO bricks are extremely precise devices . They are simple to use and allow the use of small amounts of chemoattractant solution and cell suspension, supplying by itself a triplicate test . LEGO bricks are inexpensive, fast and suitable for current diagnostic activity or for research investigations in every laboratory.

Biochem Biophys Res Commun, 2004 Oct 15, 323(2), 465 - 72
Microarray TRAP--a high-throughput assay to quantitate telomerase activity; Heller-Uszynska K et al.; Telomeric repeat amplification protocol (TRAP)--a sensitive, PCR-based assay to detect telomerase activity was quintessential to the evaluation of telomerase role in telomere maintenance, cell proliferation, tumour development, and cell immortalization . The assay, however, suffers from many limitations . The most significant are: lack of telomerase activity quantification, changes of the enzyme activity product size and/or ratio, and complex post-amplification procedures which limit the assay throughput . Here we report the development of the microarray TRAP (MTRAP) assay which combines advantages of microarray technology with a modified TRAP assay . The MTRAP was designed and optimized on rice cell suspension telomerase extract to enable telomerase specific, reliable, and linear quantification in high throughput mode, with sensitivity comparable to those of radioisotope-based TRAP assays . The MTRAP has a built-in system guaranteeing the amplification of telomerase activity products unchanged in length and/or ratio and built-in control for false negatives . Thus, our MTRAP assay provides new reliable tool for experiments requiring massive quantitation of telomerase activity .

J Microsc, 2004 Oct, 216(Pt 1), 76 - 83
Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E . coli engineered to overproduce the chemotaxis receptor Tsr; Zhang P et al.; We have recently reported electron tomographic studies of sections obtained from chemically fixed E . coli cells overproducing the 60-kDa chemotaxis receptor Tsr . Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers . Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure . The frozen-hydrated sections were generated by sectioning at -150 degrees C using a diamond knife with a 25 degrees knife angle, with nominal thicknesses ranging from 20 to 60 nm . There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.

Photochem Photobiol, 2004 Sep-Oct, 80(2), 366 - 72
A novel mitochondrial signaling pathway activated by visible-to-near infrared radiation; Karu TI et al.; The number of cells attached to glass substratum increases if HeLa cell suspension is irradiated with monochromatic visible-to-near infrared radiation before plating (the action spectrum with maxima at 619, 657, 675, 700, 740, 760, 800, 820, 840 and 860 nm) . Treating of cell suspension with sodium azide (2 x 10(-5) M), sodium nitroprusside (5 x 10(-5) M), ouabain (1 x 10(-6) M) or amiloride (1.7 x 10(-5) M) before irradiation significantly modifies the spectrum of cell attachment enhancement . A light-induced mitochondrial signaling pathway can be regulated by small ligands directly binding to the catalytic center of cytochrome c oxidase (N(3), NO) as well as by chemicals specifically binding to plasma membrane enzymes (ouabain, amiloride) . The comparative analysis of action spectra allows the conclusions that first, Cu(A) and Cu(B) chromophores of cytochrome c oxidase could be involved as photoacceptors and second, various signaling pathways (reaction channels) between cytochrome c oxidase and cell attachment regulation are at work.

Zhonghua Nan Ke Xue, 2004 Aug, 10(8), 567 - 71
{Immunomagnetic beads sorting and functional identification of human spermatogonial stem cells}; Li Y et al.; OBJECTIVE: The very nature of spermatogonial stem cells (SSC) is still poorly understood . The objective of this study is to explore the specific markers of human SSC and search for the suitable method for their isolation and functional identification . METHODS: Adults testicular cell suspensions were sorted by immunomagnetic beads method using alpha6,beta1 integrin and Thy-1 markers . The light-scattering properties and DNA Ploidy of the resultant subpopulations were analyzed by flow cytometry . The efficacies of the sorting on human SSC were evaluated by germ cell transplantation . RESULTS: (1) The alpha6+ Thy-1+ c-kit- and beta1+ Thy-1+ c-kit- cells were relatively uniform subpopulations in size and morphology, which represented about 2%-3% and 0.5%-1% of the unsorted testis cells, respectively . The analysis of light-scattering properties showed that both of the subpopulations had low side light-scattering properties . The DNA Ploidy analysis showed significant changes of these two cell subpopulations in DNA Ploidy . The percentage of diploid cells in alpha6+ Thy-1+ c-kit- cell subpopulation significantly increased to 51.2% and synthesis phase and tetraploid cells disappeared . (2) The functional evaluation showed that the SSC in the alpha6+ Thy-1+ c-kit- cells were enriched 40 times and the SSC in the beta1+ Thy-1+ c-kit- cells 20 times that of the unsorted cells . CONCLUSION: The alpha6,beta1 integrin and Thy-1 may be used for the SSC isolation as positive markers . The immunomagnetic beads sorting using alpha6,beta1 integrin and Thy-1 markers can result in significant enrichment of human SSC . It will open up a wide prospect for the researches on the biology of human SSC and the treatment of male sterility.

FEBS Lett, 2004 Sep 10, 574(1-3), 42 - 8
Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance; Droillard MJ et al.; Three of the protein kinases activated by hypoosmotic stress in Arabidopsis thaliana cell suspensions were previously characterized {FEBS, 2002, 527, 43-50} as mitogen-activated protein (MAP) kinases and two of them corresponded to Arabidopsis mitogen-activated protein kinase 6 (MPK6) (44 kDa) and MPK3 (39 kDa) . The third MAP kinase was identified here to MPK4, using a corresponding specific antibody . Like MPK6 and MPK3, MPK4 activity is clearly inhibited by apigenin and MPK4 activation by hypoosmolarity needs upstream phosphorylation events . Activation of the 3 MAP kinases, MPK3, 4 and 6, was confirmed in plantlets submitted to hypoosmotic stress . The action of a biotic signal, flagellin, was also demonstrated to induce the activations of the 3 MAP kinases . Using the mutant displaying MPK4 gene inactivation, the independence of the MPK3 and MPK6 activations towards the presence of MPK4 was demonstrated, both in hypoosmotic and flagellin signalling pathways . Although MPK4 was not activated by hyperosmolarity in cell suspensions nor in seedlings, a possible negative regulation of hyperosmolarity resistance by MPK4 is suggested, based both on phenotype and downstream gene expression studies.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Aug, 21(4), 546 - 8
{Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle}; Yao C et al.; To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage) . Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured . The DNA contents and cell cycle were analyzed by flow cytometry (FCM) . Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability . With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident . The cells exposed to SPEFs grew slower than the control . The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups . It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase . There was a significant difference in cell cycle between treated group and control group (P<0.01) . Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.

Diagn Cytopathol, 2004 Sep, 31(3), 135 - 40
Evaluation of NMP179 for the detection of squamous intraepithelial neoplasia in ThinPrep cervical slides using a combination of double immunostaining and morphometric methods of analysis; Al-Awadhi R et al.; This study investigates the potential value of the nuclear matrix protein NMP179 as a marker of abnormal squamous cells in ThinPrep slides . Forty-six cervical scrapes were collected as cell suspensions and ThinPrep slides were prepared . They were double-immunostained for NMP179 and Cytokeratin 18 (CK18), an endocervical cell marker . The method of analysis adopted for the study was designed to distinguish the abnormal squamous cells from benign epithelial cell so that the percentages of abnormal squamous cells that expressed the marker could accurately be determined . Initially, an attempt was made to identify benign and abnormal cells in the ThinPrep slides on the basis of their morphology and immunostaining patterns . Discrimination between the various types of epithelial cells was incomplete using this approach and a more precise method of discrimination between the different epithelial cell types was carried out using a combination of double immunostaining (NMP179 and CK18) and morphometry using nuclear area and nuclear cytoplasmic ratios . Once the different epithelial cell types had been identified, the specificity and sensitivity of NMP179 were determined . The optimal sensitivity (89.9%) was achieved at the N/C ratio 0.36; however, the specificity of NMP179 was very low for all N/C ratios and ranged from 38.8% to 42.2%.

J Mater Sci Mater Med, 1998 Sep, 9(9), 543 - 8
In vitro testing of surface-modified biomaterials; Leitao E et al.; The influence of surface modification treatments such as ion implantation and sputter coating on an in vitro rat bone-marrow cell culture was studied by scanning electron microscopy and X-ray microanalysis . 316 L stainless steel, Ti-6Al-4V and Ti-5Al-2.5Fe were nitrogen ion-implanted with three fluences: 1015, 1016 and 1017 ion cm-2 with an energy beam of 40 keV . Both nitrogen and carbon sputter-coated 316 L stainless steel samples were also studied . Polished 316 L stainless steel, Ti-6Al-4V, Ti-5Al-2.5Fe and ThermanoxTM were also studied, in order to give comparative information . The materials were inoculated with a droplet of cell suspension and were maintained for 3 wk . A mineralized extracellular matrix was formed on all materials except on nitrogen sputter-coated 316 L stainless steel . The morphology of the cell cultures obtained on nitrogen-ion implanted materials was similar to those obtained on the untreated materials and ThermanoxTM . The observation of the interface between the cell layer and the substrata showed the presence of calcium- and phosphorus-rich globular deposits associated with collagen fibres . A higher density of these globular deposits was observed on the ion-implanted materials.

J Mater Sci Mater Med, 1997, 8(3), 119 - 29
Dynamic blood cell contact with biomaterials: validation of a flow chamber system according to international standards; Otto M et al.; The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses . The ISO gives the standard requirements for testing biomaterials provided for implantation . Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions . The aim of the present study was to evaluate influences of different flow conditions on blood cell-biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces . PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments . The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rheometer . As reference materials we used FEP-teflon, PVC-DEHD, PU, PP and PE . Dacron and ePTFE synthetic vascular protheses were tested in a comparative way to those references . The adhesion processes were observed over a period of 40 minutes under arterial (shear stress 0.74 Pa) and venous (shear stress 0.16 Pa) flow conditions in a parallel plate flow chamber system under highly standardized conditions and laminar flow . The cells were observed with the help of inverse light microscopy . Cell behaviour was recorded and analysed in both analogue (video) and digital (imaging system) modes . Samples of the cell suspensions were obtained at regular time intervals and analysed by enzyme linked immuno sorbent assay (ELISA) to quantify LTB4 release . Irrespective of the material, approximately 3 to 4 times more PMN adhered to the biomaterial surfaces under venous flow conditions compared to the arterial . Shear intensity did not influence the running order of biomaterials with respect to cell numbers . This response in descending order at the end of the experiments was as follows: PU, PVC-DEHD, PP, PE and ePTFE . The biochemical analyses indicate that in the system used only a weak effect on LTB4 release induced by the different materials could be determined . A significant effect caused by flow conditions was not observed . Further experiments, both static as well as dynamic, must be performed for multiple, relevant parameters of haemocompatibility, for potential biomaterials as well as those currently in use in vascular prostheses.

Biochem J, 2005 Jan 1, 385(Pt 1), 217 - 23
Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress); Turner WL et al.; NADH kinase (NADHK; ATP:NADH 2'-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants . Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures . However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria . NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli . The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa . Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer . Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective K(m) values of 0.042, 0.062, 1.16, and 2.39 mM . While NADHK could phosphorylate NADH or NAD+, the specificity constant (V(max)/K(m)) for NADH was 100-fold greater than for NAD+ . The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate . PP(i) or poly-P(i) could not substitute as phospho donors . PP(i) acted as a mixed inhibitor with respect to both NADH and ATP . NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+ . This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

Microbes Infect, 2004 Sep, 6(11), 977 - 84
Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice; Indiani de Oliveira C et al.; Leishmania braziliensis is the species responsible for the majority of cases of human cutaneous leishmaniasis in Brazil . In the present study, L . braziliensis isolates from two different geographic areas in Brazil were studied by RAPD, using arbitrary primers . We also evaluated other biological features of these two isolates . We compared (a) the clinical features they initiate or not once delivered subcutaneously as stationary-phase promastigotes in the footpad of BALB/c mice; (b) the parasite load in both the footpad and the draining lymph node; (c) the cytokines present in the supernatant of cultures of the cell suspensions from the draining lymph nodes; and (d) the cell types present at the site of parasite delivery . The results show that the L . braziliensis strain from Ceara (H3227) is genotypically different from the L . braziliensis strain from Bahia (BA788) . H3227-parasitized mice developed detectable lesions, whereas BA788-parasitized mice did not . Fifteen days post parasite inoculation there was an increase in the numbers of macrophages and lymphocytes in the footpads, whatever the parasite inoculum . Parasite load at the inoculation site--namely the footpad--did not differ significantly; in draining lymph nodes, however, it increased over the period under study . Early after parasite inoculation, the cells recovered from the draining lymph nodes of BA788-parasitized mice produced higher levels of IFN-gamma, a feature coupled to a higher number of NK cells . Later, after the parasite inoculation, there was an increased content of IL-12p70 and IL-10 in the supernatant of cells recovered from the lymph nodes of H3227-parasitized mice . This comparative analysis points out that L . braziliensis isolates differing in their genomic profiles do establish different parasitic processes in BALB/c mice.

Crit Care Med, 2004 Sep, 32(9), 1904 - 9
Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock; Kirschenbaum LA et al.; OBJECTIVE: To examine the role of platelets, fibrin, and adhesion molecules in mediating neutrophil-endothelial cell interactions in septic shock . DESIGN: Controlled experiments using phase contrast microscopy to examine neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions . SETTING: University research laboratory . PATIENTS: Adult patients with septic shock and normal volunteers . INTERVENTIONS: Microslides were coated with human umbilical vein endothelial cells . Neutrophils were removed from control subjects and patients in septic shock and were perfused over endothelial cells at rates representing a range of physiologic shear stresses . In an attempt to examine the effects of fibrin deposition on neutrophil-endothelial cell interactions, neutrophils, with and without platelets, were suspended in plasma and serum was removed from patients in septic shock . In addition, blocking monoclonal antibodies against the platelet receptor P-selectin and neutrophil receptor CD11b/CD18, and a platelet glycoprotein IIb/IIIa inhibitor, were incubated with cells suspended in plasma . Phase contrast video microscopy was used to count the number of neutrophils/mm adherent to endothelial cells during cessation of flow . Neutrophil rolling velocity was calculated as the time required for neutrophils to move across a 1-mm field (mm/sec) . Leukoaggregation was defined as the number of neutrophils in aggregates (three or more nuclei) across a 1-mm field . MEASUREMENTS AND MAIN RESULTS: Normal neutrophils exposed to plasma from patients with septic shock demonstrated significant increases in aggregation and endothelial cell adherence with associated decreases in neutrophil rolling velocity . These changes were significantly enhanced in the presence of platelets and significantly attenuated in the presence of serum, which is fibrinogen depleted . Preincubation with antibodies to the surface receptors P-selectin, CD11b/CD18, and glycoprotein IIb/IIIa abrogated the changes in neutrophil aggregation, adhesion, and rolling velocity . CONCLUSIONS: These data suggest that platelets and fibrinogen play an important role in mediating neutrophil-endothelial cell adherence in septic shock.

Arch Microbiol, 2004 Oct, 182(2-3), 126 - 37 Epub 2004 Aug 31.
F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification; Seedorf H et al.; Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O(2) with H(2) at a maximal specific rate of 0.4 U (micromol/min) per mg protein with an apparent K(m) for O(2) of 30 microM . The reaction was not inhibited by cyanide . The oxidase activity was traced back to a coenzyme F(420)-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F(420)H(2) with 1 O(2) to 2 F(420) and 2 H(2)O . The apparent K(m) for F(420) was 30 microM and that for O(2) was 2 microM with a V(max) of 240 U/mg at 37 degrees C and pH 7.6, the pH optimum of the oxidase . The enzyme did not use NADH or NADPH as electron donor or H(2)O(2) as electron acceptor and was not inhibited by cyanide . The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H-X-E-X-D-X(62)-H-X(18)-D-X(60)-H . Sequence comparisons revealed that the F(420)H(2) oxidase from M . arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30-40%), which all have dioxygen reductase activity . With heterologously produced FprA from M . marburgensis it is shown that this protein is also a highly efficient F(420)H(2) oxidase and that it contains 1 FMN and 2 iron atoms . The presence of F(420)H(2) oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.

Zhong Xi Yi Jie He Xue Bao, 2004 May, 2(3), 196 - 8
{Effect of recipes replenishing qi and activating blood on cell proliferation and apoptosis in the liver of aging rats}; Yu ZY et al.; OBJECTIVE: To observe the effect of recipes replenishing qi and activating blood on cell proliferation and apoptosis in the liver of natural aging rats . METHODS: Natural aging rats were under administration of recipes replenishing qi or activating blood for 4 months . The liver of the rats was prepared into cell suspension for determination of cell proliferation and apoptosis with PI-staining and flow cytometer . RESULTS: (1) Compared with those of the young rats, the cells in G(0)-G1 phase in the liver tissue of aging rats were increased (P< 0.01), and apoptosis cells were increased (P< 0.01), while the cells in S and G2-M phases were decreased (P< 0.01) . (2) Compared with those of the aging rats, the cells in G(0)-G1 phase in the liver tissue of aging rats administered recipes replenishing qi or activating blood were decreased (P< 0.01), and it was more obvious in activating blood group than in replenishing qi group (P< 0.01); the cells in S and G2-M phases were increased (P< 0.01) and there was no significant difference between the activating blood group and the replenishing qi group (P> 0.05) . (3) The apoptosis cells in replenishing qi or activating blood group were decreased significantly (P< 0.01), and the effect of replenishing qi was better than that of activating blood (P< 0.01) . CONCLUSION: (1) Cell proliferation is decreased and apoptosis is increased in the liver tissue of natural aging rats . (2) Recipes replenishing qi or activating blood can accelerate cell proliferation in the liver tissue of natural aging rats, and the effect of activating blood was slightly stronger than that of replenishing qi . (3) Recipes replenishing qi or activating blood can inhibit cell apoptosis in the liver tissue of natural aging rats, and the effect of replenishing qi was better than that of activating blood.

Acta Derm Venereol, 2004, 84(4), 265 - 70
Functional characterization of beta1-integrin-positive epidermal cell populations; van Rossum MM et al.; Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide . We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ . Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments . Image analysis was used to determine growth characteristics of the colonies . We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation . The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation . Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different . Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Inflamm Res, 2004 Aug, 53 Suppl 2, S105 - 8 Epub 2004 Aug 10.
Adverse reactions to drugs: in vitro studies with isolated cells; Ennis M et al.; OBJECTIVE AND DESIGN: Drug-induced adverse reactions can be allergic or pseudoallergic in nature, in this study the histamine releasing ability of 4 radiographic contrast media and 2 opioid analgesics was tested on a variety of mast cell containing cell suspensions . MATERIAL: Mast cell containing cell suspensions were obtained from porcine lung, liver, kidneys and heart, as well as rat lung and rat peritoneal lavage . TREATMENT: Cells were incubated for 10 min with Angiographin (amidotrizoate), Hexabrix (ioxaglate), Rayvist (ioglycate) or Telebrix (ioxithalamate) all 10-500 microl/ml or with levomethadone (0.1-5 mM) or pethidine (0-10 mM) . METHODS: Histamine was measured using an automated fluorometric method and percentage histamine release calculated . RESULTS: All agents caused histamine release from porcine cells and rat lung cells . However, rat peritoneal mast cells were refractory to the action of pethidine . Cardiac cells were the most sensitive to the radiographic contrast media but the least sensitive to the opioid analgesics . CONCLUSIONS: The data indicate that mast cells isolated from animal models can provide an indication of those agents likely to induce an adverse pseudoallergic reaction . However, these data should be used together with those obtained from human mast cells, in vivo animal experiments as well as studies using human volunteers and clinical trials as advocated in the Marburg Model .

J Exp Bot, 2004 Oct, 55(406), 2235 - 40 Epub 2004 Aug 27.
Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine; Tesniere C et al.; Although grape berries have been classified as non-climacteric fruits, ongoing studies on grape ethylene signalling challenge the role of ethylene in their ripening . One of the significant molecular changes in berries is the up-regulation of ADH (alcohol dehydrogenase, EC 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels . This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions . The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene receptors . Treatment of cell suspensions with 2-chloroethylphosphonic acid (2-CEPA), an ethylene-releasing compound, also resulted in a significant increase in ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes in VvADH2 expression . All these results associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that the ethylene transduction pathway and anaerobic stress could be, in part, involved in the regulation of VvADH2 expression in ripening berries and cell suspensions . These data open new aspects of the expression control of a ripening-related gene in a non-climacteric fruit.

Anticancer Res, 2004 Jul-Aug, 24(4), 2225 - 30
Antineoplastic effect of immunostimulatory DNA (CpG-ODN) in a murine C57-BL6/MB-49 transitional cell carcinoma model; Hegele A et al.; BACKGROUND: Intravesical BCG installation is the standard of care in the prophylaxis of recurrent intermediate and high-risk transitional cell carcinoma (TCC), but its mode of action has not yet been elucidated . However, a Th-1 biased immune response is postulated Cell culture and animal models demonstrated the efficacy of synthetic CpG-oligodeoxynucleotides (ODN) as inducer and adjuvant for a strong Th1-response . The purpose of our study was to evaluate the antineoplastic effect of locally administered CpG ODN in a subcutaneous murine bladder cancer model . MATERIALS AND METHODS: A subcutaneous murine TCC model was established in female C57/BL6 mice using the corresponding syngeneic MB49 TCC cell line . Three groups of 5 animals received a cell suspension, standardized for 1x10(6) cells/50 microl, injected s.c . into the right and left flank . Group I received 10 nmol of CpG-ODN only into the right cell depot . Group II received 10 nmol of GpC ODN . Group III served as untreated control and received only PBS . The animals were examined at various time points after injection until sacrifice on day 14 . Tumor or scar tissue were excised, weighed and examined histopathologically (HE-stain) . RESULTS: Tumor sizes and weights showed no side differences . The average tumor weight on day 14 was 171 mg (SD +/- 8.9), 110 mg (SD +/- 19.2) and 18 mg (SD +/- 6.1), respectively, in groups III, II and I (p<0.05) . Histopathology revealed solid vital epithelial tumors in group III and reduced vital tumor mass with central necrosis and moderate mononuclear infiltration in group II . Group I showed almost complete tumor necrosis and a considerable mononuclear inflammatory response . CONCLUSION: Immunostimulatory DNA has promising antineoplastic activity in a murine subcutaneous TCC-model . The histological findings suggest an immunologically-mediated mode of action . Further investigations are necessary to elucidate the immunological response.

Acta Physiol Scand, 2004 Sep, 182(1), 53 - 62
Isolation and receptor profiling of ileal enterochromaffin cells; Schafermeyer A et al.; AIM AND METHODS: Enterochromaffin (EC) cells, interspersed throughout the gastrointestinal mucosa, provide most of the serotonin of the body and control intestinal motility, secretion and absorption . We purified EC cells from the rat ileum by a combination of elutriation and density gradient centrifugation in order to characterize the function of this important cell type . RESULTS: Immunostaining showed that there were 84% serotonin-positive cells in the highly enriched EC fraction as compared with 12% in unfractionated cells, yielding a approximately sevenfold enrichment . Serotonin measurements in the cell suspensions indicated a seven to 14-fold enrichment . Presence of alpha- and beta-adrenoreceptor isoforms, muscarinic M3 and gama-aminobutyric acid (GABA)-A receptors was confirmed by RT-PCR and cytochemistry . Increased expression of VMAT-1 and GABA-A mRNA was also shown by quantitative TaqMan PCR using EC cell RNA . Serotonin release in isolated EC cells was stimulated by noradrenaline, and to a smaller extent, by carbachol, while GABA addition was without effect . CONCLUSION: Our data provide a basis for a new approach to characterize receptors on this unique cell type.

Biofizika, 2004 May-Jun, 49(3), 511 - 8
{Analysis of the kinetics of Ca2+ signals in Ehrlich ascites tumor cells upon the inhibition of the mitochondrial Na+/Ca2+ exchanger}; Novel fluoro- and hydroxyl-containing jasmonate derivatives as highly efficient elicitors in suspension cultures of Taxus chinensis; Shanghai Key Lab . of Chemical Biology, Institute of Pesticides and Pharmaceuticals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, ChinaTo develop more effective abiotic elicitors for cell suspension cultures of T . chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol . Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T . chinensis cell cultures.

Shi Yan Sheng Wu Xue Bao, 2004 Jun, 37(3), 237 - 40
{Influence of substrates coated on cultured dorsal root ganglion cells}; Guo L et al.; Dorsal root ganglia from rat embryos were digested with trypsin to form single cell suspension . The cells were seeded on culture plates coated respectingly with poly-L-lysin (PLL), laminin (LN), PLL combined with LN, or collagen type I (CoI), then cultured in NB1 media . The cell survival and neurite outgrowth of cultured dorsal root ganglion cells were observed by phase-contrast microscopy . The results showed that the dorsal root ganglion neurons grown on PLL combined with LN were dispersively distributed at high survival rate, and those cells grown on CoI were clustered with thicker and longer neurites . It is suggested that the cell growth pattern could be influenced by different substances coated on the plates, and PLL combined with LN may provide a better substrum to culture and study single neuron soma and neurite.

Ophthalmologe, 2004 Sep, 101(9), 886 - 94
{Transplantation of retinal pigment pithelium (RPE) following CNV removal in patients with AMD . Techniques, results, outlook}; Bindewald A et al.; Neovascular age-related macular degeneration (AMD) has become the leading cause for severe visual loss in all industrialized nations . Surgical excision of choroidal neovascularizations (CNV) is technically feasible but invariably associated with inadvertent removal of corresponding retinal pigment epithelium (RPE) and subsequent atrophy of the choriocapillaris, with the latter two layers being a prerequisite for normal photoreceptor function . To cover the RPE defect both heterologous and homologous RPE cell suspensions have been injected into the subretinal space . The lack of functional improvement has been attributed to various factors including RPE cell dedifferentiation, failure of adherence to Bruch's membrane as well as development of a regular RPE cell monolayer . Therefore, techniques for translocating intact autologous RPE cell sheets have been sought and preservation of foveal neurosensory functions has recently been successfully demonstrated . Besides translocation of a full-thickness RPE/Bruch's membrane/choroid patch outside the macular area, superfluous choroidal tissue may be ablated intraocularly using an excimer laser prior to translocation . Besides recent pharmacological approaches including anti-VEGF agents, these surgical developments open new perspectives for patients with neovascular AMD.

Plant Cell, 2004 Sep, 16(9), 2364 - 79 Epub 2004 Aug 17.
CycD1, a putative G1 cyclin from Antirrhinum majus, accelerates the cell cycle in cultured tobacco BY-2 cells by enhancing both G1/S entry and progression through S and G2 phases; Koroleva OA et al.; A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells . To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2 . Green fluorescent protein:CycD1 is located in the nucleus throughout interphase . Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries . We examined the effect of induced expression at different stages of the cell cycle . Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis . Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins . The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression . Continuous expression of CycD1 led to moderate increases in growth rate . Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression . This indicates that D cyclin function may have diverged between plants and animals.

Eur J Cancer, 2004 Sep, 40(13), 1987 - 92
The happy destiny of frozen haematopoietic stem cells: from immature stem cells to mature applications; de Vries EG et al.; Forty years ago, van Putten described in the European Journal of Cancer (see this issue) quantitative studies on the optimal storage techniques of mouse and monkey bone marrow suspensions . Survival of the animals after irradiation following injection with stored bone marrow cell suspensions was the endpoint . He observed some species differences, but based on the data obtained considered a careful trial of the glycerol-polyvinylpyrrolide (PVP) combination for storage of marrow in man was indicated . In spite of this, dimethyl sulphoxide has become the 'standard' cryopreservant for human marrow stem cells . Over the last 40 years, there has been a tremendous increase in knowledge about haematopoietic stem cells and their use in the clinic . Haematopoietic stem cells are now known to travel between the bone marrow and peripheral blood and are the best-characterised adult stem cells . These cells are currently widely used for transplantations in the clinic and are obtained from a wide variety of sources . These include the bone marrow, peripheral blood, cord blood, autologous as well as allogeneic stem cells from related or unrelated donors . Increasingly, data has become available that adult haematopoietic stem cells can generate differentiated cells belonging to other cell types, a process called "developmental plasticity" . Thus, they may contribute to non-haematopoietic tissue repair in multiple organ systems . This has created a whole new potential therapeutic armamentarium for the application of haematopoietic stem cells outside of the area of malignancies and haematopoietic disorders.

Physiol Res, 2004, 53(4), 439 - 47
Effects of exogenous donor of nitric oxide - sodium nitroprusside on energy production of rat reticulocytes; Maletic SD et al.; The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated . Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM) . SNP decreased total and coupled, but increased uncoupled oxygen consumption . This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation . Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP . Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished . Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production . In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels . However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level) . ATP/ADP ratio and adenylate energy charge level were significantly decreased . In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes . These alterations were accompanied with instability of energy status.

Methods Mol Biol, 2005, 286, 61 - 78
Stable transformation of plant cells by particle bombardment/biolistics; Kikkert JR et al.; Particle bombardment, or biolistics, is a commonly used method for genetic transformation of plants and other organisms . Millions of DNA-coated metal particles are shot at target cells or tissues using a biolistic device or gene gun . The DNA elutes off the particles that lodge inside the cells, and a portion may be stably incorporated in the host chromosomes . A protocol for the generation of transgenic grapevines via biolistic transformation of embryogenic cell suspension cultures is detailed in this chapter . In a typical experiment, transient gene expression averaged nearly 8000 "hits" per bombarded plate . Five months after bombardment, there were nearly five putative transgenic embryos per bombarded plate . About half of the embryos were regenerated into confirmed transgenic plants . The basic bombardment procedures described are applicable to a wide range of plant genotypes, especially those for which embryogenic cell cultures are available . All users of particle bombardment technology will find numerous useful tips to maximize the success of transformation.

Yao Xue Xue Bao, 2004 Apr, 39(4), 305 - 8
{Effects of fungal elicitor on inophyllums production in suspension cultured cells of Calophyllum inophyllum L.}; Luo HL et al.; AIM: To investigate the effects of fungal elicitors on inophyllums production in suspension cultured cell of Calophyllum inophyllum Linn . METHODS: The pathogen of leaf spot disease of C . inophyllum L . was isolated and prepared as fungal elicitor . The fungal elicitor was added to the medium with different concentrations and culture period . Their effects on biomass and inophyllums content of the suspension of cultured cells were studied . RESULTS: The optimum effects of S-I fungal elicitor concentrations on inophyllums content was 60 mg GE x L(-1) . Adding the fungi elicitor into the cell suspension culture system at stationary phase (being cultured for 18 days) resulted in a highest inophyllum content of 59.174 mg x L(-1) at the 3rd day with 27% higher than control . Fungal elicitor treatment promoted the inophyllums accumulation in medium . CONCLUSION: Adding the Stagonospora curtisii (Berk.) Sacc . to the medium was effective approaches to enhance inophyllums yield in the suspension of C . inophyllum L culture cell.

Hum Exp Toxicol, 2004 Jun, 23(6), 307 - 16
Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures; Hewitt NJ et al.; Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)) . CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional . Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions . In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis . However, this effect was substrate and species dependent and was not always the same in fresh and CP cells . CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation . CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells . Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture . Species-specific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures . In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.

Ophthalmologe, 2004 Sep, 101(9), 882 - 5
{Transplantation of iris pigment epithelium}; Thumann G et al.; Transplantation of iris pigment epithelial (IPE) cells to the subretinal space has been attempted as a therapeutic modality for the treatment of age-related macular degeneration (AMD) . IPE cells are used because autologous cells are readily available and because IPE and RPE cells share a common embryonic origin, possess the capacity of transdifferentiation into other ocular cells, and share common morphological and functional characteristics . Once the technique of IPE cell transplantation was established in an animal mode, several clinical studies analyzed the behavior of IPE cell suspensions transplanted to the subretinal space of patients with AMD following surgical membrane extraction . In our experience, as well as that of other investigators, transplantation of IPE cells to the subretinal space of AMD patients prevents the recurrence of the subretinal neovascularization and stabilizes but does not improve visual acuity . Since IPE cells transplanted as a cell suspension do not appear to form a cell monolayer in the subretinal space, the transplantation of preformed IPE or RPE cell monolayers is being investigated as the development of an functional cell monolayer is mandatory if functional success, i.e., recovery of vision in AMD patients, is the ultimate goal of IPE cell transplantation.

J Interferon Cytokine Res, 2004 Jul, 24(7), 416 - 27
Colony-stimulating factor-1 promotes clonogenic growth of normal murine colonic crypt epithelial cells in vitro; Ramsay RG et al.; The intestinal epithelium is a continuously renewing tissue . In the colon, stem cells are maintained at the base of highly organized crypts, where they undergo asymmetric division and give rise to daughter cells that proliferate and migrate up the crypt as they differentiate, then become senescent and are finally shed into the intestinal lumen . The growth factor requirements of fetal and prenatal colon cells for colony formation and that influence the establishment of cell lines from Immorto-mouse (Charles River, Wilmington, MA) transgenic embryos were explored . Single cell suspensions were isolated and cultured in a large range of growth factor combinations and conditions to determine their growth properties in soft agar . We report an important advance in the culture of mouse colonocytes by using macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) . A substantial proportion of colonies grown under low oxygen tension in the presence of CSF-1 and GM-CSF express intestinal epithelial A33 antigen, have the expected gene expression profile, including c-fms and transcription factor c-myb, and show an appropriate epithelial cell morphology and undetectable CD45 . Confocal microscopy on isolated crypts displays basolateral expression of c-Fms and E-cadherin on most epithelial cells . Fetal colon cultures from the Immorto-mouse with CSF-1 produced rapid outgrowth and readily established cell lines, in contrast to cultures without CSF-1 . These observations have implications for the understanding of colon epithelial development and recovery following cytotoxic damage as well as providing a basis for the observation that some colon (and other epithelial) tumor cells respond to CSF-1 and GM-CSF.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1245 - 50
Growth behavior in plant cell cultures based on emissions detected by a multisensor array; Komaraiah P et al.; The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described . The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor . The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions . By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M . citrifolia . The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1183 - 91
Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations; Ma N et al.; Pluronic F-68 (PF-68) is routinely used as a shear-protection additive in mammalian cell cultures . However, most previous studies of its shear protection mechanisms have typically been qualitative in nature and have not covered a wide range of PF-68 and cell concentrations . In this study, interactions between air bubbles along with the associated cell damage were investigated using the novel adenovirus-producing cell line PER.C6, a human embryonic retinoblast transfected with the adenovirus type 5 E1 gene . A wide range of PF-68 and cell concentrations (approximately 3 orders of magnitude) were used in these studies . At low PF-68 concentrations (0.001 g/L), cells had a very high affinity for bubbles, indicated by a more than 10-fold increase in cell concentration in the foam layer liquid versus the bulk liquid . At high PF-68 concentrations ( approximately 3 g/L), however, the cell concentration in the foam layer liquid was only approximately 40% of that in the bulk cell suspension . The number of cells associated with each bubble decreased from approximately 1000 cells at 0.001 g/L PF-68 to approximately 120 cells at 3 g/L PF-68 . Despite the lower cell affinity for bubbles at a high PF-68 concentration, at high cell concentrations (10(7) cells/mL and 1 g/L PF-68) significant cell entrapment occurred in the foam layer, on the order of 1000 cells/bubble . For the cells carried by the bubbles, quantitative cell damage data revealed that the probability of cell death from bubble rupture was independent of bulk cell concentration but was affected by PF-68 concentration . These quantitative studies further indicated that even at a low PF-68 concentration of 0.03 g/L, approximately 30% of the attached cells were killed during the bubble rupture process . At the same time, at low PF-68 concentration (<0.1 g/L), significant cell death occurred prior to bubble rupture . On average, a bubble disrupted more cells in the bulk liquid and/or foam layer than during rupture . For both mechanisms, the number of cells damaged by each bubble increased with decreasing PF-68 concentration and increasing bulk cell concentration.

Appl Environ Microbiol, 2004 Aug, 70(8), 4532 - 7
Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1; Thibodeau J et al.; A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1 . The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively . The reductive dehalogenase was purified 12.7-fold to apparent homogeneity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa . Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN . A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor . Several polychlorophenols were dechlorinated at the meta and para positions . The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM . Six internal tryptic peptides were sequenced by mass spectrometry . One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences . This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase . The corresponding ORF (named cprA5) in D . frappieri PCP-1 was cloned and sequenced . The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion . The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases . This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.

Tree Physiol, 2004 Oct, 24(10), 1181 - 6
High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce; Helmersson A et al.; Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death . In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes . We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis . Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid . We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines . There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin . The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants . We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

Protein Expr Purif, 2004 Sep, 37(1), 89 - 96
Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants; Gao J et al.; Coagulation factor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many "wound sealants" which are proposed for use or currently in use as effective hemostatic agents, sealants, and tissue adhesives in surgery . After activation by alpha-thrombin cleavage, coagulation factor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent cross-linking of the alpha- and gamma-chains of linear fibrin to form homopolymers, which can quickly stop bleeding . We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants . Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins . The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII . Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and cross-linking activity in the presence of linear fibrin . The expression of factor XIII A-domain was not affected by plant leaf position.

J Biol Chem, 2004 Oct 1, 279(40), 41758 - 66 Epub 2004 Jul 29.
Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana; Boudsocq M et al.; Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension . Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress . One of them was identified to AtMPK6 but the others remained to be identified . They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group . The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate . Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins . Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays . Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling . In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2 . The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed.

Curr Opin Chem Biol, 2004 Aug, 8(4), 392 - 8
Flow cytometry for high-throughput, high-content screening; Edwards BS et al.; Flow cytometry is a mature platform for quantitative multi-parameter measurement of cell fluorescence . Recent innovations allow up to 30-fold faster serial processing of bulk cell samples . Homogeneous discrimination of free and cell-bound fluorescent probe eliminates wash steps to streamline sample processing . Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries . Novel bead-based assay systems allow studies of real-time interactions between solubilized receptors, ligands and molecular signaling components that recapitulate and extend measurements in intact cells . These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing and drug discovery.

Genome, 2004 Aug, 47(4), 680 - 8
Regeneration of somatic hybrids in relation to the nuclear and cytoplasmic genomes of wheat and Setaria italica; Xiang F et al.; Somatic hybridization via PEG (Polyethylene 6000)-mediated protoplast fusion was achieved between two different wheat culture lines (Triticum aestivum L., "Jinan"177, T1 and T2) and Setaria italica (L.) P . Beauv . The T1 recipient originated from non-regenerable long-term cell suspensions, while T2 was derived from embryogenic calli with a high regeneration capacity . Donor protoplasts were obtained from embryogenic calli of S . italica (S) (with low regeneration capacity) irradiated with different doses of ultraviolet light . Twenty-three putative hybrid cell lines were produced in fusion combinations with the donor protoplasts treated with UV light for 30 s (combination I) and 1 min (combination II), but only one (from combination II) differentiated into green plants . Three cell lines from combination I and five cell lines from combination II possessed the nuclear genomes of T1, T2, and S . italica as revealed by cytological, isozyme, RAPD, and 5S rDNA spacer sequence analyses . Genomic in situ hybridization (GISH) analysis showed that most hybrid cell lines had 22-36 wheat chromosomes, 0-2 S . italica chromosomes, and 1-6 wheat - S . italica recombinant chromosomes, whereas the regenerable cell line had 44-56 wheat chromosomes and 3-6 recombinant chromosomes, but no intact S . italica chromosomes . RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted in all hybrid cell lines and recombined in most hybrid cell lines . These results indicate that the regeneration of hybrid plants involves not only the integration of S . italica nuclear and organellar DNA, but also the genome complementation of T1 and T2.

Cancer Surv, 1998, 31, 29 - 47
Malignancy associated papillomaviruses and morphology of human bladder cancer; Oliver RT et al.; Animal studies in rabbit and cattle have clearly demonstrated the contribution of host genetics, chemical carcinogens and immunosuppression to the conversion of papillomavirus induced benign regressing warts into malignant cancers . More significant is the role of vaccination both with whole tumour cell suspensions with whole virus and viral proteins, particularly L2 molecules, in causing progressing warts to regress . Early results in small scale studies of HPV16 E6/E7 vaccine in patients with cervical cancer have provided evidence that tumour regression can be induced in human papillomavirus induced tumours . These observations provided added impetus for more research to firm up the increasing, but still principally anecdotal, evidence that papillomaviruses may be involved in the pathogenesis of bladder cancer . Studies of carcinomas arising in cattle after BBV 4 infection show absence of fully infectious virus in the majority of tumours, though the tumours have persistent E7, E8 and LCR sequences . As this is all that is required for transformation, it may require in vitro molecular studies in human bladder cancer screening for such elements before final proof of involvement is confirmed . However, even before this is achieved, given the success in animal models of whole tumour cell vaccines, serious thought should be given to how to develop protocols for study of crude tumour cell vaccines in vivo . Such studies would need in vitro assays to seek evidence for specific antitumour immunity, focusing on studies of tumour infiltrating lymphocytes and their T cell receptor polymorphisms.

Phytochemistry, 2004 Jul, 65(13), 1911 - 7
Salt-induced lipid changes in Catharanthus roseus cultured cell suspensions; Elkahoui S et al.; Salt treatment strongly affected cell growth by decreasing dry weight . Exposure of Catharanthus roseus cell suspensions to increasing salinity significantly enhanced total lipid (TL) content . The observed increase is mainly due to high level of phospholipids (PL) . Hundred mM NaCl treatment increased phospholipid species phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it reduced glycolipid ones monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) but not sulfoquinovosyldiacylglycerol (SQDG) . Moreover, fatty acid composition was clearly modified when cells were cultured in the presence of 100 mM NaCl, whereas only few changes occurred at 50 mM . Salt treatment decreased palmitic acid (16:0) level and increased that of linolenic acid (18:2) . Such effect was observed in phospholipid species PC and PE and in glycolipid DGDG . Double bond index (DBI) was enhanced more than 2-fold in fatty acids of either glycolipids or phospholipids from cells submitted to 100 mM NaCl . Free sterol content was also significantly enhanced, especially at 100 mM NaCl, whereas free sterols/phospholipids (St/PL) ratio was slightly decreased . All these salt-induced changes in membrane lipids suggest an increase in membrane fluidity of C . roseus cells.

Lasers Med Sci, 2004, 19(1), 33 - 6 Epub 2004 Jun 23.
Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma; Knoll T et al.; New approaches in the treatment of transitional cell carcinoma (TCC) are using gene therapy to influence the disease at the genetic level . Technical advances in genomics, the availability of tissue-specific gene promoters and other developments have made this approach more realistic . Transporting the gene into the target cell is still the major problem . Several transfection techniques have been introduced . Transfection of naked DNA is one of the simplest to perform but transfection rates have been very poor . We investigated the influence of laser energy on transfection efficacy in urothelial cancer cells in vitro with two types of medical lasers . A suspension of human transitional cancer cells (UM-UC3; 3.5 million cells/ml) was mixed with 200 microg of plasmid DNA (pEGFP-N1) . Two types of laser energy, neodymium:YAG (Nd:YAG) and holmium:YAG (Ho:YAG), were applied to the cell suspension in different energy settings . Twenty four hours after treatment, transfection rates were measured with FACS analysis . Energy setting parameters that determine the efficacy of laser were investigated . The significance of different transfection rates was estimated with the student's t-test . We demonstrated that the Nd:YAG laser was not suitable for achieving significant transfection of the reporter gene to the cells . In contrast, the Ho:YAG laser produced satisfactory transfection rates . There was an increase in transfection with increasing frequency of laser pulses, from 16% with 2 Hz up to 40% with 10 Hz (p < 0.0005) . Pulse frequency was therefore stabilised at 10 Hz . Pulse energy (mJ) showed the same dependency: a transfection rate of 18.3% was achieved with 1,000 mJ and 53.8% with 2,000 mJ (p > 0.0005) . Additionally, we investigated the impact of total pulse number (imp) with different pulse energies . At 1,000 mJ, a transfection rate of 18.3% was estimated with 200 imp and 48.56% with 750 imp, (p < 0.0005) . At 2,000 mJ, a transfection rate of 53.8% was achieved with 200 imp and 58.26% with 500 imp . The optimal laser setting observed in this experiment was 10 Hz, 2,000 mJ and 500 imp . This study indicates that the efficacy of naked DNA delivery into TCC in vitro is improvable by application of Ho:YAG laser energy . The Nd:YAG laser did not increase transfection rates in our model . Our results with the Ho:YAG laser are encouraging for further studies to optimise DNA delivery . As TCC tissue is relatively easy to access, this method could become an effective and minimally invasive procedure in urothelial cancer treatment.

Phytochemistry, 2004 Jun, 65(11), 1575 - 88
Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry; Zhang C et al.; Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium . CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185 . ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans . Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS . The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS . The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site . Good agreement was found with the one glycan previously analyzed by (1)H NMR . This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.

Phytochemistry, 2004 Jun, 65(12), 1829 - 38
A proteomic analysis of plant programmed cell death; Swidzinski JA et al.; Programmed cell death (PCD) is an active cellular suicide that occurs in animals and plants throughout development and in response to both abiotic and biotic stresses . In contrast to animals, little is known about the molecular machinery that regulates plant PCD . We have previously identified transcriptomic changes associated with heat- and senescence-induced PCD in an Arabidopsis cell suspension culture {Plant J . 30 (2002) 431} . However, since plant PCD is also likely to involve elements that are regulated post-transcriptionally, we have undertaken a proteomic analysis in the Arabidopsis system . We identified 11 proteins that increased in abundance relative to total protein in both treatments despite extensive degradation of other proteins . We argue that some of these proteins are maintained during PCD and may therefore have specific functions in the PCD pathway . The increased abundance of several antioxidant proteins as well as a measured increase in free Fe2+ content of the cells indicates an oxidative stress in this system . Several mitochondrial proteins were identified, confirming the importance of this organelle during PCD . We also identified an extracellular glycoprotein that may function in the transmission of a 'death signal' from cell to cell . Putative roles for the identified proteins are presented.

Phytochemistry, 2004 Jun, 65(12), 1693 - 707
The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions; Brugiere S et al.; The development of mitochondria and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting membranes . The aim of the present work was: (1) to enhance our understanding of the biochemical machinery of mitochondrial membranes and (2) to test the versatility of the procedure developed for the identification of the hydrophobic proteome of the chloroplast envelope {Molecular and Cellular Proteomics 2 (2003) 325-345} . A proteomic analysis was performed, to provide the most exhaustive view of the protein repertoire of these membranes . For this purpose, highly purified mitochondria were prepared from Arabidopsis cultured cells and membrane proteins were extracted . To get a more exhaustive array of membrane proteins from Arabidopsis mitochondria, from the most to the less hydrophobic ones, various extraction procedures (chloroform/methanol extraction, alkaline or saline treatments) were applied . LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of more than 110 proteins . The identification of these proteins is discussed with respect to their mitochondrial localization, their physicochemical properties and their implications in the metabolism of mitochondria . In order to provide a new overview of the biochemical machinery of the plant mitochondria, proteins identified during this work were compared to the lists of proteins identified during previous proteomic analyses performed on plant and algae mitochondria (Arabidopsis, pea, Chlamydomonas, rice, etc.) . A total of 502 proteins are listed . About 40% of the 114 proteins identified during this work were not identified during previous proteomic studies performed on mitochondria.

Biotechnol Lett, 2004 May, 26(10), 793 - 8
Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine; El-Sayed M et al.; Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in the accumulation of catharanthine, tabersonine and condylocarpine . Condylocarpine is not an intermediate in the pathway to catharanthine or tabersonine when it is fed to the cultures . The results support the hypothesis that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine.

Clin Cancer Res, 2004 Jul 15, 10(14), 4699 - 708
Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses; Avigan D et al.; PURPOSE: Dendritic cells (DCs) are potent antigen-presenting cells that are uniquely capable of inducing tumor-specific immune responses . We have conducted a Phase I trial in which patients with metastatic breast and renal cancer were treated with a vaccine prepared by fusing autologous tumor and DCs . EXPERIMENTAL DESIGN: Accessible tumor tissue was disrupted into single cell suspensions . Autologous DCs were prepared from adherent peripheral blood mononuclear cells that were obtained by leukapheresis and cultured in granulocyte macrophage colony-stimulating factor, interleukin 4, and autologous plasma . Tumor cells and DCs were cocultured in the presence of polyethylene glycol to generate the fusions . Fusion cells were quantified by determining the percentage of cells that coexpress tumor and DC markers . Patients were vaccinated with fusion cells at 3-week intervals and assessed weekly for toxicity, and tumor response was assessed at 1, 3, and 6 months after completion of vaccination . RESULTS: The vaccine was generated for 32 patients . Twenty-three patients were vaccinated with 1 x 10(5) to 4 x 10(6) fusion cells . Fusion cells coexpressed tumor and DC antigens and stimulated allogeneic T-cell proliferation . There was no significant treatment-related toxicity and no clinical evidence of autoimmunity . In a subset of patients, vaccination resulted in an increased percentage of CD4 and CD8+ T cells expressing intracellular IFN-gamma in response to in vitro exposure to tumor lysate . Two patients with breast cancer exhibited disease regressions, including a near complete response of a large chest wall mass . Five patients with renal carcinoma and one patient with breast cancer had disease stabilization . CONCLUSIONS: Our findings demonstrate that fusion cell vaccination of patients with metastatic breast and renal cancer is a feasible, nontoxic approach associated with the induction of immunological and clinical antitumor responses.

J Heart Lung Transplant, 2004 Jul, 23(7), 881 - 8
Acute effects of direct cell implantation into the heart: a pressure-volume study to analyze cardiac function; Ishida M et al.; BACKGROUND: To safely implant cells into the myocardium, we must establish a volume that prevents compromising cardiac performance . We studied pressure-volume (PV) to investigate the adverse effects of direct cell implantation in the acute phase . METHODS: We used 21 minipigs . In the normal heart model, we studied PV by measuring various parameters (including end-systolic pressure, end-systolic elastance, dp/dtmax, end-diastolic volume, and time constant of isovolumetric left ventricular pressure fall {Tau}) . We injected solutions into the left ventricular free wall (15 cm(2)) . Sampling points were at baseline and after injection of saline (Group I, n = 4) or of blood (Group II, n = 4) at volumes of 1 ml and 10 ml up to 30 minutes after injection . In Group II, we injected additional blood (10 ml) 4 times . In the ischemic heart model, 1 month after ligating the left anterior descending artery, we injected 1 ml saline (Group III, n = 4), bone marrow mononuclear cells (10(8) cells/1 ml; Group IV, n = 4), or bone marrow stromal cells (10(8) cells/1 ml; Group V, n = 3) . We studied PV before and after injection . RESULTS: In Group I, we found no significant changes in parameters . In Group II, end-diastolic volume after 10-ml injection (24.4 +/- 3.6 ml) was smaller than end-diastolic volume at baseline (29.5 +/- 5.8 ml, p < 0.01) . Tau after 10-ml injection (39.4 +/- 5.3 msec) was greater than at baseline (35.6 +/- 4.0 msec, p < 0.01) . One pig died of ventricular fibrillation after a 20-ml injection of blood . We observed no detrimental effects in Groups III, IV, and V . CONCLUSIONS: More than 10 ml cell suspension compromised diastolic function . We safely performed direct injection of bone marrow cells (1 x 10(8)/1 ml).

Colloids Surf B Biointerfaces, 2004 Apr 15, 34(4), 221 - 30
Cell-cell contact and membrane spreading in an ultrasound trap; Coakley WT et al.; An ultrasonic standing wave trap {Langmuir 19 (2003) 3635} in which the morphologies of 2-D latex-microparticle aggregates, forming a pressure node plane, were characterised has been applied here to different cell suspensions with increasing order of specificity of cross-linking molecule, i.e . polylysine with chondrocytes; wheat germ agglutinin (WGA) with erythrocytes and surface receptors on neural cells . The outcome of initial cell-cell contact, i.e . whether the cells stuck at the point of contact (collision efficiency = 1) or rolled around each other (collision efficiency = 0), was monitored in situ by video-microscopy . The perimeter fractal dimensions (FD) of 2-D hexagonally symmetric, closely packed aggregates of control erythrocytes and chondrocytes were 1.16 and 1.18, respectively while those for the dendrititc aggregates formed initially by erythrocytes in 0.5microg/ml WGA and chondrocytes in 20 microg/ml polylysine were 1.49 and 1.66 . The FDs for control and molecularly cross-linked cells were typical of reaction-limited aggregation (RLA) and transport diffusion-limited aggregation (DLA), respectively . The FDs of the aggregates of cross-linked cells decreased with time to give more closely packed aggregates without clear hexagonal symmetry . Suspensions of neural cells formed dendritic aggregates . Spreading of inter-cellular membrane contact area occurred over 15 min for both erythrocyte and neural cell dendritic aggregates . The potential of the technique to characterise and control the progression of cell adhesion in suspension away from solid substrata is discussed.

Indian J Exp Biol, 2004 Jun, 42(6), 616 - 9
Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats; Narasimhan S et al.; Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins . Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production . Berberine was synthesized and released continuously into the liquid medium . Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis . On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth . Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D . IAA and IBA were not favourable for cell growth and berberine synthesis.

Am J Physiol Lung Cell Mol Physiol, 2004 Nov, 287(5), L902 - 10 Epub 2004 Jul 16.
Mechanisms of early pulmonary neutrophil sequestration in ventilator-induced lung injury in mice; Choudhury S et al.; Polymorphonuclear leukocytes (PMN) play an important role in ventilator-induced lung injury (VILI), but the mechanisms of pulmonary PMN recruitment, particularly early intravascular PMN sequestration during VILI, have not been elucidated . We investigated the physiological and molecular mechanisms of pulmonary PMN sequestration in an in vivo mouse model of VILI . Anesthetized C57/BL6 mice were ventilated for 1 h with high tidal volume (injurious ventilation), low tidal volume and high positive end-expiratory pressure (protective ventilation), or normal tidal volume (control ventilation) . Pulmonary PMN sequestration analyzed by flow cytometry of lung cell suspensions was substantially enhanced in injurious ventilation compared with protective and control ventilation, preceding development of physiological signs of lung injury . Anesthetized, spontaneously breathing mice with continuous positive airway pressure demonstrated that raised alveolar pressure alone does not induce PMN entrapment . In vitro leukocyte deformability assay indicated stiffening of circulating leukocytes in injurious ventilation compared with control ventilation . PMN sequestration in injurious ventilation was markedly inhibited by administration of anti-L-selectin antibody, but not by anti-CD18 antibody . These results suggest that mechanical ventilatory stress initiates pulmonary PMN sequestration early in the course of VILI, and this phenomenon is associated with stretch-induced inflammatory events leading to PMN stiffening and mediated by L-selectin-dependent but CD18-independent mechanisms.

Cell Tissue Bank, 2001, 2(4), 241 - 247
Application of Banked Autologous Mucosal Cells to Graft Large Intraoral Mucosal Defects; Grossman N et al.; Purpose: This study was aimed to investigate the conditions that affect the generation of autologous cultured mucosal grafts (CMG) to promote their clinical application for large intraoral defects, following surgical excision of mucosal pathology . Specific parameters studied were the effect of patient's age and cell banking on the in vitro development of CMG, and on their clinical performance in vivo.Patients and methods: Twelve patients (10 M, 2F; mean age of 50.7+/-14.3 years) with intraoral mucosal pathologies were included in this study . Autologous mucosal cells derived from 0.2-0.5 cm(2) biopsies, taken from clinically normal oral mucosa, were cultured in vitro and banked, as cell suspensions, in liquid nitrogen . Two to three weeks before the scheduled surgery, multilayered CMG were developed according to the anticipated area of the excised tissue (8-100 cm(2)), from banked or proliferating autologous cells (for 7/12 and 5/12 patients, respectively) . Following excision, CMG sheets were placed on the mucosal defects and anchored to the adjacent tissue with sutures, which were removed a week later.Results: Both the production of CMG, and their clinical performance were unaffected significantly by patient's age . Three weeks postoperatively, the grafted sites were smooth and keratinized, without infection or scar contraction, with complete and comparable healing among the two groups, treated with banked and non-banked cells.Conclusion: This study indicates that CMG is a proper dressing for large intraoral mucosal defects of various etiologies, for a large range of patients' age, using either banked or non-banked cells.

Cell Tissue Bank, 2002, 3(1), 3 - 10
The role of quality control in a skin bank: tissue viability determination; Alotto D et al.; New surgical procedures requiring viable skin have increased rapidly over the last few years . The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability . Various methods, both qualitative and quantitative, are currently used . Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting . Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability . Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing . Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes . The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength . It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension . However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks . Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals . In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation . In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability . Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.

Zhonghua Bing Li Xue Za Zhi, 2004 Jun, 33(3), 198 - 202
{Distinction between benign and malignant pheochromocytomas}; Liu TH et al.; OBJECTIVES: To investigate the differences in morphology, immunohistochemistry, DNA ploidy status, LOH and MSI of 11q13 and 1p between benign and malignant pheochromocytomas, and to find the marker or markers useful in distinction between benign and malignant pheochromocytoma or for predicting the malignant potential of this tumor . METHODS: Twenty-two cases of clinically documented benign and malignant pheochromocytomas from the files of Peking Union Medical College Hospital were analyzed . Aside from histological study, Ki-67, p53, CgA, S-100, PCNA and survivin immunohistochemistry studies were performed . DNA ploidy status was assessed by flow cytometry on cell suspensions prepared from formalin-fixed, paraffin-embedded sections . Twelve tumors (7 benign and 5 malignant) with paired normal tissues were microdissected . Tumor and normal tissue DNA were extracted . The obtained DNAs and 8 microsatellite markers related to 11q13 and 1q were subjected to PCR amplification for analysis of LOH and MSI . RESULTS: None of the tumors showed atypical mitosis, only 1 malignant tumor had a mitotic count > 1/10 HPF (2.3/10 HPF) . Two malignant tumors exhibited confluent necrosis . Ki-67 index was low in benign tumors (average 0.73%), and high in malignant tumors (average 2.4%) . The difference of Ki-67 index between benign and malignant tumors was statistically significant . DNA ploidy status did not correlate with malignancy . Although LOH and/or MSI of 11q13 and 1p were observed in several tumors, a statistically significant difference could not be reached due to the small number of tumors analyzed . CONCLUSION: Only Ki-67 index (> 3%) is an useful marker for distinguishing benign from malignant or for predicting the malignant potential of pheochromocytoma.

Int J Mol Med, 2004 Aug, 14(2), 295 - 9
Production and efficacy of a dendritic cell-based therapeutic vaccine for murine chronic hepatitis B virus carrierer; Akbar SM et al.; Recently a new field of immunological research and clinical application of vaccines for therapeutic purposes (vaccine therapy) has been developed for treating several chronic viral infections including chronic hepatitis B virus (HBV) infection . Administration of vaccine containing hepatitis B surface antigen (HBsAg) for 1 year has resulted in negative HBsAg and development of antibody to HBsAg (anti-HBs) in some, but not in all, HBV transgenic mouse (HBV-Tg) . In order to develop more potent regimen of vaccine therapy for chronic HBV carrier, we prepared a dendritic cell (DC)-based therapeutic vaccine and evaluated their therapeutic potential in HBV-Tg . DCs were isolated from single cell suspensions of murine spleen cells by collagenase digestion, density centrifugation and depletion of lymphocytes . Spleen DCs were cultured with HBsAg (100 microg) for 24 h to produce HBsAg-pulsed DCs . HBV-Tg expressing HBsAg and HBV DNA in the sera were randomly assigned to receive either HBsAg-pulsed DCs (n = 20) or unpulsed DC (n = 20) or vaccine containing HBsAg (n = 39) or complete Freund's adjuvant (n = 20) or left untreated (n = 20) . Only two intraperitoneal injections of HBsAg-pulsed DCs resulted in negative HBsAg and production of anti-HBs in the sera in all HBV-Tg (n = 20) . However, administration of un-pulsed DCs (n = 20) or vaccine containing HBsAg (n = 39) or only complete Freund's adjuvant did not induce negative HBsAg or production of anti-HBs in any HBV-Tg within 6 months of therapy commencement . Taken together, this study showed that HBsAg-pulsed DCs represent a highly potent therapeutic vaccine for chronic HBV infection and inspire optimism of using this vaccine in clinical conditions . A clinical trial of HBsAg-pulsed DC in patients with chronic hepatitis B is warranted.

J Immunol Methods, 2004 Jun, 289(1-2), 179 - 90
Human tonsillar tissue block cultures differ from autologous tonsillar cell suspension cultures in lymphocyte subset activation and cytokine gene expression; Giger B et al.; Lymphoid tissues cultured either as tissue blocks or as cell suspensions are used to study the behaviour of immune cells within their habitat . The preservation of tissue structures in tissue blocks, which is considered to be a major advantage, has been poorly defined . We characterised the morphological evolution of tissue cultures from human palatine tonsils and compared their lymphocyte subsets and the constitutive cytokine gene expression to those in autologous tonsillar single-cell suspension cultures over time, and after adding cyclosporin A (CsA) to mimic the situation in individuals treated with immunosuppressive drugs . Density and morphology of follicles were conserved up to 4 days, during which tissue cultures exhibited similar cell viability as suspension cultures, but a significantly less frequent increase of CD95 expression in T cells, smaller variation of the proportion of CD4(+) cells and better CD21(+)/CD23(-) B-cell survival . Treatment with cyclosporin A at higher concentrations resulted in superior histologic preservation of lymphoid tissue structures and seemed to further prevent the expression of CD95 by CD3(+) cells and the activation in tissue culture of CD21(+) cells . Constitutive gene expression levels of the stromal cytokines interleukin (IL)-1beta and interleukin-6 in tissue culture were significantly higher than those in suspension cultures . These results suggest that tonsillar tissue cultures preserve their structure only for a limited time, during which they more closely reflect processes in vivo, including a state of iatrogenic immunosuppression, than do their cell suspension counterparts.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 261 - 6
Sustained glycolytic oscillations--no need for cyanide; Poulsen AK et al.; Using fluorescence spectroscopy we detected long trains of macroscopic oscillations in the glycolytic pathway, in whole cell suspensions of Saccharomyces cerevisiae, without addition of cyanide . Such oscillations may be induced if argon or another inert gas is bubbled through the yeast cell suspension . This supports that the synchronizing agent is a volatile compound secreted by the yeast cells, e.g . CO2 and/or acetaldehyde . Our results show that the rate of acetaldehyde removal is not a crucial parameter to the synchronization of the yeast cells . The sample cell was connected to a membrane inlet mass spectrometer (MIMS) for online determination of extracellular non-polar compounds . Oscillations in the secretion of CO2 were detected using the MIMS.

Plant Physiol, 2004 Jul, 135(3), 1378 - 87 Epub 2004 Jul 09.
AtOPT6 transports glutathione derivatives and is induced by primisulfuron; Cagnac O et al.; The oligopeptide transporter (OPT) family contains nine members in Arabidopsis . While there is some evidence that AtOPTs mediate the uptake of tetra- and pentapeptides, OPT homologs in rice (Oryza sativa; OsGT1) and Indian mustard (Brassica juncea; BjGT1) have been described as transporters of glutathione derivatives . This study investigates the possibility that two members of the AtOPT family, AtOPT6 and AtOPT7, may also transport glutathione and its conjugates . Complementation of the hgt1met1 yeast double mutant by plant homologs of the yeast glutathione transporter HGT1 (AtOPT6, AtOPT7, OsGT1, BjGT1) did not restore the growth phenotype, unlike complementation by HGT1 . By contrast, complementation by AtOPT6 restored growth of the hgt1 yeast mutant on a medium containing reduced (GSH) or oxidized glutathione as the sole sulfur source and induced uptake of {3H}GSH, whereas complementation by AtOPT7 did not . In these conditions, AtOPT6-dependent GSH uptake in yeast was mediated by a high affinity (Km = 400 microm) and a low affinity (Km = 5 mm) phase . It was strongly competed for by an excess oxidized glutathione and glutathione-N-ethylmaleimide conjugate . Growth assays of yeasts in the presence of cadmium (Cd) suggested that AtOPT6 may transport Cd and Cd/GSH conjugate . Reporter gene experiments showed that AtOPT6 is mainly expressed in dividing areas of the plant (cambium, areas of lateral root initiation) . RNA blots on cell suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that AtOPT6 expression is strongly induced by primisulfuron and, to a lesser extent, by abscisic acid but not by Cd . Altogether, the data show that the substrate specificity and the physiological functions of AtOPT members may be diverse . In addition to peptide transport, AtOPT6 is able to transport glutathione derivatives and metal complexes, and may be involved in stress resistance.

Eur J Neurosci, 2004 Jul, 20(1), 92 - 100
Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue; Binder W et al.; Stress induces analgesia by mechanisms within and outside the brain . Here we show that the sympathetic nervous system is an essential trigger of intrinsic opioid analgesia within peripheral injured tissue . Noradrenaline, injected directly into inflamed hind paws of male Wistar rats, produced dose-dependent antinociception, reversible by alpha(1)-, alpha(2)- and beta(2)-antagonists . alpha(1)-, alpha(2)- and beta(2)-adrenergic receptors were demonstrated on beta-endorphin-containing immune cells and noradrenaline induced adrenergic receptor-specific release of beta-endorphin from immune cell suspensions . This antinociceptive effect of noradrenaline was reversed by micro - and delta-opioid antagonists as well as by anti-beta-endorphin . Stress-induced peripheral analgesia was abolished by chemical sympathectomy and by adrenergic antagonists . These findings indicate that sympathetic neuron-derived noradrenaline stimulates adrenergic receptors on inflammatory cells to release beta-endorphin, which induces analgesia via activation of peripheral opioid receptors.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Jul, 16(7), 409 - 12
{Protective effect of specific antibody in serum of convalescent patient with SARS}; Li Y et al.; OBJECTIVE: To investigate inhibitory effect of serum severe acute respiratory syndrome (SARS) -specific antibodies from convalescent patients after half an year of onset on SARS-CoV-mediated cytopathic response . METHODS: SARS-CoV immunoglobulin G (IgG) antibody was determinated by enzyme linked immunoadsorbent assay (ELISA) . Twelve serum samples from convalescent patients, diluted by 1:8 with maintenance medium, were mixed with the three dilution supernatants of SARS-CoV . SARS-CoV were isolated, cultured and identified by the Guangzhou Institute of Respiratory Disease, and cultured with Vero E6 cell suspension . The extent of cytopathic response was observed . RESULTS: The absorbance (A) value of SARS-CoV IgG antibody ranged from 0.81 to 2.06 in patients after half an year of SARS onset, and form 0.79 to 2.01 in patients before half an year of SARS onset . The extent of cytopathic response was decreased by more than 25% in all 12 convalescent patients, as compared with control serum . CONCLUSION: The A value of SARS-CoV IgG antibody in serum of convalescent patients tended to elevate in half an year after SARS onset . SARS-CoV IgG antibody could inhibit SARS-CoV-mediated cytopathic response, indicating it might be one of protective antibodies.

Magn Reson Imaging, 2004 Jul, 22(6), 843 - 50
MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents; Funovics MA et al.; Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development . In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate . MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination . The relaxivity of the conjugates was 24.1-29.1 mM(-1) s(-1), with 1.8 to 2.1 antibody molecules per nanoparticle . A panel of cultured melanoma and mammary cell lines was used for testing . The cells were incubated with the particles at 16-32 microg Fe/ml in culture medium for 3 h at 37 degrees C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination . All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T(2) signal intensity at 1.5 T . The changes in 1/T(2) were between 1.5 and 4.6 s(-1) and correlated with the amount of cell-bound iron (R = 0.92) . The relaxivity of cell-bound MION increased to 55.9 +/- 10.4 mM(-1) s(-1) . TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis . In conclusion, we obtained receptor-specific T(2) MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens . This concept can potentially be expanded to a large number of targets and to in vivo applications.

J Surg Res, 2004 Aug, 120(2), 189 - 94
Small intestinal submucosa does not promote PAIII tumor growth in Lobund-Wistar rats; Hodde JP et al.; BACKGROUND: Site-specific remodeling and angiogenesis are two observations associated with the use of small intestinal submucosa (SIS) as a tissue repair graft . Its angiogenic capacity has raised questions concerning its effect on tumor growth and metastasis in clinical tumor resection cases . The effect of SIS on the ability of neoplastic (prostate adenocarcinoma) cells to establish, grow, and metastasize was examined in Lobund-Wistar (L-W) rats . MATERIALS AND METHODS: In one study, SIS, expanded polytetrafluoroethylene (ePTFE), or human cadaveric dermis was placed in a subcutaneous pocket on the flank of L-W rats and immediately inoculated with PA-III cell suspension . Tumors were allowed to establish and metastasize for 5 weeks prior to sacrifice . Rate of tumor growth, tumor weight, and frequency of lung metastases were assessed . In a second study, SIS was placed in a resected tumor bed and tumors were allowed to recur . Rate of tumor growth, tumor weight, and frequency of lung metastases were assessed after 3 weeks . RESULTS: ePTFE hastened the rate of formation of palpable tumors compared to controls and other materials; cadaveric dermis and SIS did not . No differences between materials were noted in final tumor weight nor in the frequency of metastasis to the lungs . Following surgical tumor resection, residual tumor cells led to recurrence of same-site tumors in all animals, but in the defects augmented with SIS, the tumors were significantly smaller than those which regrew in the resected, unaugmented group . CONCLUSIONS: This study demonstrates that SIS does not enhance tumor establishment, growth, or metastasis in de novo tumors . Furthermore, SIS appears to reduce the rate of tumor growth, but not metastasis, when applied in direct contact with a residual tumor bed in a rat model of prostate-related tumors.

Eur J Biochem, 2004 Jul, 271(14), 2976 - 83
Cloning and expression of a tomato cDNA encoding a methyl jasmonate cleaving esterase; Stuhlfelder C et al.; Jasmonic acid and its methyl ester are ubiquitous plant signalling compounds necessary for the regulation of growth and development, as well as for the response of plants to environmental stress factors . To date, it is not clear whether methyl jasmonate itself acts as a signal or if its conversion to jasmonic acid is mandatory prior to the induction of a defense response . We have cloned a cDNA, encoding a methyl jasmonate-cleaving enzyme, from tomato cell suspension cultures . Sequence analysis revealed significant similarity to plant esterases and to (S)-hydroxynitrile lyases with an alpha/beta-hydrolase fold structure . The coding sequence was heterologously expressed in Escherichia coli and purified in a catalytically active form . Transcript levels, as well as enzymatic activity, were determined in different tomato tissues . High transcript levels and enzyme activities were found in roots and flowers, while the mRNA level and activity were low in stems and leaves . Moreover, when tested in methyl jasmonate- and elicitor-treated cell suspension cultures, transcript levels were found to decrease, indicating that this particular enzyme might be a regulator of jasmonate signalling.

Ultramicroscopy, 2004 Aug, 100(3-4), 171 - 8
Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy; Lesniewska E et al.; Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view . The objective of this work was to further describe this mechanism . Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions . The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24h after elicitation . In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells . After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains . These expansions were not observed on untreated grapevine cells . The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties . The elasticity is diminished in UV-treated cells . In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response . These results demonstrate cell wall strengthening by UV stress.

Brain Res, 2004 Jul 23, 1015(1-2), 50 - 6
Intracerebral transplantation of carotid body in rats with transient middle cerebral artery occlusion; Yu G et al.; Recent laboratory and clinical studies demonstrate therapeutic efficacy of intracerebral transplantation of carotid body (CB) in Parkinson's disease, possibly through secretion of neurotrophic factors . Here, we examined the role of CB in experimental stroke . In the first experiment, we hypothesized that removal of CB would exacerbate cerebral infarction and stroke-related behavioral deficits . Eight-week-old, male Sprague-Dawley rats were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB . We used the stroke model of temporary middle cerebral artery occlusion . The ipsilateral CB was removed in animals assigned to treatment group exposed to stroke with surgically removed CB . Behavioral tests, using the elevated body swing test, were conducted at days 1-3 after surgery . Cerebral infarction was visualized by TTC staining on day 3 post-surgery . The data revealed no significant differences in behavioral deficits and infarct volumes between the two groups . In the second experiment, CB cell suspension grafts or control adult tissue grafts were intracerebally transplanted into the ischemic penumbra immediately (within 1 h) after stroke surgery . The results revealed significant reduction of behavioral deficits and infarct volumes, accompanied by increased levels of neurotrophic factors, as detected by ELISA, in transplanted ischemic striatum collected from CB-grafted stroke animals . These observations suggest that surgical resection of CB in the periphery did not alter stroke pathology; however, CB when made available in the CNS, via intracerebral transplantation, could protect against stroke possibly through the synergistic release of neurotrophic factors . The present study extends the use of CB as efficacious graft source for transplantation.

Cryo Letters, 2004 May-Jun, 25(3), 213 - 7
Cryopreservation of Helianthus tuberosus cell suspension cultures: the effect of preculture treatments on cytoskeletal proteins and transglutaminase activity; Harris W et al.; Helianthus tuberosus cell suspension cultures were subjected to cryopreservation 24h preculture treatments with 0.5M sucrose or mannitol . Extracts were assayed for transglutaminase activity and the level of alpha-tubulin tyrosination . There was a significant reduction (compared with the non-precultured controls) in transglutaminase activity and alpha-tubulin tyrosination state after mannitol preculture treatment, whereas sucrose preculture treatment produced no significant effect . The results suggest that reduced levels of transglutaminase activity and alpha-tubulin tyrosination are associated with a lack of post-thaw recovery observed following mannitol preculture treatment of cell culture suspensions . These activities may represent useful molecular markers of the success of preculture treatments in cryopreservation protocols.

Plant Cell Physiol, 2004 Jun, 45(6), 672 - 83
Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana; Shimaoka T et al.; A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole . To date, few proteins involved in these activities have been identified at the molecular level . In this study, proteomic analysis was used to identify new tonoplast proteins . A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high . Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles . No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum . The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS . Using this approach, it was possible to identify 163 proteins . These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast . There were also a number of proteins for which a function has not yet been deduced.

Exp Cell Res, 2004 Jul 15, 297(2), 593 - 606
Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture; Hirobe T et al.; Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF) . Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion) . GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures . Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF . Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes . Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes . These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.

Toxicology, 2004 Aug 5, 200(2-3), 193 - 203
The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine; Carvalho M et al.; In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity . The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes . Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h . To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds) . The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST) . MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST . In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities . For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation . Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA . The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes . Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.

Br J Haematol, 2004 Jul, 126(1), 111 - 9
A mouse model to study organ homing behaviour of haemopoietic progenitor cells reveals high selectivity but low efficiency of multipotent progenitors to home into haemopoietic organs; Henschler R et al.; To study the homing behaviour of an enriched multipotent primitive haemopoietic progenitor cell (HPC) population in mice, undifferentiated murine factor-dependent multipotent HPCs (FDCP-mix), stably transfected with the green fluorescence protein gene, were intravenously injected into congenic mice . After 2 or 24 h, cell suspensions were prepared from bone marrow, spleen, lung, liver, muscle, colon, kidney, brain or blood of the mice and analysed by flow cytometry . Using direct quantifiable determination of total HPC numbers homed per organ and a method to estimate the degree of organ contamination by HPC that were present in blood vessels within the organs before preparation, the highest absolute numbers of HPC were detected in the liver and lungs at 2 h but this was sharply decreased at 24 h, whereas HPC selectively accumulated in the bone marrow and spleen at 24 h after transplantation . Only a few HPC were detected in other organs . The seeding efficiency of homed FDCP-mix HPC to the bone marrow and spleen was approximately 1.5% and ranged between that of primary whole bone marrow cells and lineage-depleted freshly isolated bone marrow cells . Pretreatment of HPC with inhibitors of signal transduction indicated that short-term homing of multipotent HPC into haemopoietic organs is an active process requiring co-ordinated intracellular signalling through Rho family small GTPases and protein kinases . Thus, short-term homing of FDCP-mix HPC into haemopoietic organs is of low efficiency but high selectivity, and provides a system to analyse the mechanisms and manipulation of primitive HPC which saves large numbers of donor animals.

J Neurosci Methods, 2004 Aug 15, 137(1), 25 - 35
Dissociation of retinal ganglion cells without enzymes; Hayashida Y et al.; We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections . Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter . Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter . The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated . After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve . These cells showed no obvious morphological degeneration for several days of culture . Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.

Transplant Proc, 2004 May, 36(4), 1166 - 8
Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas; Poliakova L et al.; Side population (SP) cells defined by their ability to efflux Hoechst dye 33342 (Hst), demonstrate functional stem cell capabilities in adult murine tissues and may represent organ-specific stem cells . We examined adult human (Hu) and rhesus macaque (Rh) pancreatic tissue for the presence of SP cells . METHODS: Hu cadaver (n = 4) and Rh donor (n = 5) pancreata were dispersed with collagenase and separated by density gradient centrifugation to relatively enrich fractions for islet, ductal, and acinar tissue in human and islet and nonislet tissue in Rh . Single cell suspensions were incubated with varying Hst concentrations to determine optimal conditions for SP cell analysis . Cellular heterogeneity was assessed using a panel of monoclonal antibodies positive for hematopoietic and/or endothelial cells . RESULTS: Hu SP cells comprised approximately 0.12%, 0.08%, and 0.45% of the gated populations for Hu islet, ductal, and acinar fractions respectively . In Rh, 5.5% and 3.7% of the islet and nonislet fractions were identified as SP cells . FACS analysis of Hu pancreas-derived SP cells indicated that greater than 95% were CD45(-), and only 6% were CD34(+)CD45(-) . A similar phenotype was detected in Rh pancreas-derived SP cell populations: greater than 70% were CD45(-) and less than 2% were endothelial lineage positive . CONCLUSIONS: SP cells are found in both islet- and nonislet-enriched fractions of the adult Hu and Rh pancreas . The majority of pancreatic SPs are CD45(-) and CD34(-), suggesting nonhematopoietic lineage . Further preclinical study is needed to establish the phenotype and functional role of adult tissue-specific versus tissue-resident stem cells.

Plant Physiol Biochem, 2004 May, 42(5), 403 - 9
Enhancement of photosynthetic O2 evolution in Chlorella vulgaris under high light and increased CO2 concentration as a sign of acclimation to phosphate deficiency; Kozlowska-Szerenos B et al.; The photosynthetic oxygen evolution of Chlorella vulgaris (Beijer.) cells taken from phosphate-deficient (-P) and control cultures was measured during 8 days of culture growth . Under inorganic carbon concentration (50 microM) in the measuring cell suspension and irradiance (150 micromol m(-2) s(-1)), the same as during culture growth, there were no marked differences in the photosynthetic O2 evolution rate between the -P cells and the controls . The much slower growth of -P cultures indicated that the utilization of absorbed photosynthetically active radiation (PAR) in the CO2 assimilation and biomass production were in -P cells less efficient than in the controls . Alga cells under the phosphorus stress utilized more of the absorbed PAR in the nitrate reduction than the control cells . However, under conditions of more efficient CO2 supply (inorganic carbon concentration 150 microM, introducing of exogenous carbonic anhydrase to the measuring cell suspension) and under increased irradiance (500 micromol m(-2) s(-1)), the photosynthetic O2 evolution in -P cells reached a higher rate than in the controls . The results suggest that in -P cells the restricted CO2 availability limits the total photosynthetic process . But under conditions more favorable for the CO2 uptake and under high irradiance, the -P cells may reveal a higher photosynthetic oxygen evolution rate than the controls . It is concluded that an increased potential activity of the photosynthetic light energy absorption and conversion in the C . vulgaris cells from -P cultures is a sign of acclimation to phosphorus stress by a sun-type like adaptation response of the photosynthetic apparatus .

Cell Transplant, 2004, 13(3), 273 - 82
Reassessment of caspase inhibition to augment grafted dopamine neuron survival; Marchionini DM et al.; One experimental therapy for Parkinson's disease (PD) is the transplantation of embryonic ventral mesencephalic tissue . Unfortunately, up to 95% of grafted neurons die, many via apoptosis . Activated caspases play a key role in execution of the apoptotic pathway; therefore, exposure to caspase inhibitors may provide an effective intervention strategy for protection against apoptotic cell death . In the present study we examined the efficacy of two different caspase inhibitors, caspase-1 inhibitor Ac-YVAD-CMK and caspase-3 inhibitor Ac-DEVD-CMK, to augment mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival in culture and following implantation into the denervated striatum of rats . We report that treatment with Ac-YVAD-CMK provided partial but nonsignificant protection for TH-ir neurons against serum withdrawal in mesencephalic cultures plated at low density, while neither caspase inhibitor promoted TH-ir neuron survival in higher density cultures, simulating graft density . We demonstrate that plating procedures (full well vs . microislands) and cell density directly affect the degree of insult experienced by TH-ir neurons following serum withdrawal . This varying degree of insult directly impacts whether caspase inhibition will augment TH-ir neuron survival . Our grafting experiments demonstrate that Ac-YVAD-CMK does not augment grafted TH-ir neuron survival when added to mesencephalic cell suspensions prior to grafting or to mesencephalic reaggregates for 3 days in vitro prior to transplantation . These experiments provide further evidence of the failure of these caspase inhibitors to augment TH-ir neuron survival . Furthermore, we suggest that cell culture paradigms used to model grafting paradigms must more closely approximate the cell densities of mesencephalic grafts to effectively screen potential augmentative treatments.

Cell Transplant, 2004, 13(3), 263 - 71
Liposomal formulations of tacrolimus and rapamycin increase graft survival and fiber outgrowth of dopaminergic grafts; Alemdar AY et al.; The immunosuppressive drugs tacrolimus (TAC) and rapamycin (RAPA) have both been found to have neuroprotective effects on dopaminergic neurons . The purpose of the present study was to investigate whether liposomal formulations of these drugs administered directly into the brain improve cell survival and fiber outgrowth . Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal rat ventral mesencephalic cells and randomly divided to one of four groups . Group 1 received a transplant containing cells only; group 2 received a cell suspension containing 0.68 microM liposomal RAPA (LRAPA); group 3 received a cell suspension containing 2.0 microM liposomal TAC (LTAC); and group 4 received a cell suspension containing a liposomal formulation of both 0.68 microM RAPA and 2.0 microM TAC (LRAPATAC) . Rats were sacrificed after 6 weeks, and cell survival and fiber outgrowth were assessed using tyrosine hydroxylase (TH) immunohistochemistry . The animals receiving a cell suspension containing either LTAC or LRAPATAC were found to have significantly more surviving TH-immunoreactive (TH-ir) cells than the control group receiving cells only . The group receiving LTAC had significantly longer fibers, the group receiving LRAPA had significantly more fibers close to the graft, and the group receiving LRAPATAC had significantly more fibers at all distances . This study shows the feasibility of using liposomal formulations of neuroimmunophilins directly in the brain at the time of implantation to improve graft survival and fiber outgrowth . Furthermore, we have shown that the combination of LTAC and LRAPA has a synergistic effect . These compounds may play an important role in optimizing graft survival and host reinnervation in cell-mediated brain repair strategies for the treatment of neurological conditions.

Cell Transplant, 2004, 13(3), 245 - 52
The effects of transforming growth factor-beta2 on dopaminergic graft survival; Macauley SL et al.; Dopaminergic cell transplantation is a promising therapeutic approach for the treatment of Parkinson's disease, the potential of which is limited due to poor survival and low dopamine content within engrafted tissue . In this study, the ability of transforming growth factor-beta2 (TGF-beta2) to influence transplant survival was evaluated . Cell suspensions containing fetal rat ventral mesencephalon (VM) cells were incubated prior to surgery with vehicle (DPBS), varying concentrations of TGF-beta2 (5-1000 ng/ml), or a pan-specific antibody against TGF-beta (1D11, 100 ng/ml) . VM cell suspensions (200,000 cells) were unilaterally implanted into the striatum of adult Sprague-Dawley rats (n = 5-11 animals/group) . Following a 3-week survival period, small but viable VM grafts containing tyrosine hydroxylase-positive (TH+) neurons and fibers were present in all animals . Addition of TGF-beta2 resulted in a steep, bell-shaped dose-response curve with a significant effect on TH+/dopamine cell survival . At 50 ng/ml TGF-beta2, the number of surviving dopamine neurons was increased twofold compared with controls . Addition of TGF-beta2 or 1D11 did not significantly influence graft volume . Further studies, possibly in combination with other neurotrophic factors, need to be performed to obtain a greater understanding of the effects of TGF-beta on dopamine neurons and fetal VM cell engraftment.

Pathol Oncol Res, 2004, 10(2), 109 - 16 Epub 2004 Jun 09.
The pattern of cytokine gene expression in human colorectal carcinoma; Csiszar A et al.; Systemic and local cytokine environment may modulate the immunogenicity of colorectal cancer cells, and affect anti-tumor immune functions of tumor-infiltrating lymphocytes . We therefore investigated cytokine mRNA expression patterns in tumors and peripheral blood mononuclear cells (PBMC) from patients with colorectal adenocarcinoma . IL-2, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-4, IL-6, IL-8, IL-10 and IL-1 beta mRNAs in single cell suspension of freshly isolated colorectal cancer tissue were studied by RT-PCR . Frequencies of cytokine gene expression were compared to those in normal colonic mucosa from tumor patients . The frequencies of IL-2, IFN-gamma, IL-4 and IL-10 gene expression were also determined in peripheral blood mononuclear cells from patients with colorectal adenocarcinoma and compared to those of healthy individuals . Tumor samples were more frequently positive for IFN-gamma, IL-2, TNF-alpha and IL-10 gene expression than normal mucosa (p=0.0001, p=0.0118, p=0.001 and p<0.0001, respectively) . Frequencies of IL-2 and TNF-alpha gene expressions were significantly higher in tumors with a diameter <5 cm, than in those with a diameter >5 cm . The genes for IL-6, IL-1 beta and IL-8 were commonly expressed in both tumor tissue and normal colonic mucosa . IFN-gamma transcripts were detected in more PBMC samples from patients with colorectal cancer than those from normal controls (p=0.0449) . Thus, colorectal cancer tissue is characterized by a specific pattern of cytokine gene expression . It is likely that multiple interactions between pro- and anti-inflammatory cytokines regulate tumor growth and the functional activity of tumor-infiltrating lymphocytes.

Int J Biochem Cell Biol, 2004 Sep, 36(9), 1848 - 59
Obligatory involvement of CD26/dipeptidyl peptidase IV in the activation of the antiretroviral tripeptide glycylprolylglycinamide (GPG-NH(2)); Balzarini J et al.; GPG-NH2 and G-NH2 are highly selective antiretroviral agents in cell culture, and both compounds inhibit HIV replication in CEM cell cultures to an equal extent (50% effective concentration: approximately 30 microM) . The lymphocyte surface glycoprotein marker CD26, which is identical to dipeptidyl peptidase IV, efficiently converted GPG-NH2 to G-NH2 releasing the dipeptide GP-OH . The closely related QPG-NH2 derivative was also inhibitory to HIV, presumably by the dipeptidyl peptidase IV (DPP IV)-catalyzed release of G-NH2 . In contrast, the cyclic pQPG-NH2 derivative in which the glutamine at the amino terminal position of QPG-NH2 was replaced by pyroglutamine and which is resistant to cleavage by purified CD26, was devoid of antiviral activity . CD26 is abundantly expressed on a variety of HIV target cells and is also present in serum of bovine, murine and human origin . The CD26/DPP IV enzymatic activity in serum and in cell suspensions could be efficiently inhibited by the CD26/DPP IV inhibitor L-isoleucinepyrrolidine (IlePyr) with 50% inhibitory concentrations ranging between 20 and 100 microM . When combined in HIV-1-infected cell cultures, IlePyr and Diprotin A (DP-A), another CD26/DPP IV inhibitor, abrogated the antiviral activity of GPG-NH2 but not of G-NH2 . Therefore, it was concluded that the anti-HIV drug GPG-NH2 is not active as such, but rather behaves as a prodrug that must be obligatorily cleaved by CD26/DPP IV to G-NH2 to exert its antiretroviral activity . This is the first demonstration of a lymphocyte activation/differentiation marker (i.e . CD26) that plays a direct regulatory and indispensable role in the eventual antiretroviral activity of small synthetic molecules such as the antiretroviral (pro)drug GPG-NH2 .

Exp Eye Res, 2004 Jul, 79(1), 41 - 9
Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures; Kim HS et al.; Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction . Explant and single cell systems are currently used for human corneal epithelial cultivation . This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns . Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer . The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43) . BrdU labeling was performed to identify the label-retaining cells . Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems . The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age . Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures . The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures . The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture . Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems . BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days . In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells . The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems . Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

FEBS Lett, 2004 Jun 4, 567(2-3), 197 - 202
Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells; Kaminaga Y et al.; Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to various glucosides . The glucosylation efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures prior to curcumin administration . Two cDNAs encoding UDP-glucosyltransferases (CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C . roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases . The sequence identity between their deduced amino acid sequences was 27% . The expression of both genes was up-regulated by addition of MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than that of CaUGT2 . The corresponding cDNAs were expressed in Escherichia coli as fusion proteins with maltose-binding protein . The recombinant CaUGT1 exhibited no glucosylation activity with either curcumin or curcumin monoglucoside as substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin monoglucoside from curcumin and also conversion of curcumin monoglucoside to curcumin diglucoside . The use of the recombinant CaUGT2 may provide a useful new route for the production of curcumin glucosides.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Jul 25, 807(1), 129 - 37
Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles; Plieva FM et al.; Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm) . Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm . Cryogel porosity depended on cryo-polymerization conditions . More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls . The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h . Chromatographic behavior of E . coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.

J Comp Neurol, 2004 Jun 21, 474(2), 246 - 60
Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells; Berglund CM et al.; An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods . Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days . Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter . Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes . Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality . Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture . Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes . The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells .

Biotechnol Bioeng, 2004 Jun 30, 86(7), 817 - 26
Preparation of single cells from aggregated Taxus suspension cultures for population analysis; Naill MC et al.; A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry . Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions . The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures . The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase . High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines . In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines . Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures) . In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures . The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate . The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism .

J Agric Food Chem, 2004 Jun 2, 52(11), 3467 - 72
Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera); Krasnow MN et al.; Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-cancer effects when consumed in the human diet . Glycosylation of stilbenes increases their solubility and may make them more easily absorbed by the intestine . We have found an activity in extracts of cultured cells of Vitis vinifera (cv . Gamay Freaux) that glucosylates the stilbene resveratrol to form piceid . The Km for UDP-Glucose was 1.2 mM, and the Km for resveratrol was 0.06 mM, values similar to those of other phenolic glucosyltransferases . We investigated the resveratrol glucosylating activity of the enzyme extracted from cells grown under different light treatments (dark, visible light, light + ultraviolet (UVC) radiation) and found the activity to be unaffected or slightly reduced . In contrast, UVC light strongly stimulated extractable quercetin glucosyltransferase activity . These results, combined with analysis of phenolic compounds extracted from the differently treated cells, suggest that the resveratrol glucosyltransferase is distinct from the glucosyltransferase(s) active on other phenolics.

J Agric Food Chem, 2004 Jun 2, 52(11), 3337 - 44
Herbicidal pyridyl derivatives of aminomethylene-bisphosphonic acid inhibit plant glutamine synthetase; Obojska A et al.; A series of aminomethylene-bisphosphonic acid derivatives, previously synthesized and shown to be endowed with herbicidal properties, were evaluated as potential inhibitors of plant glutamine synthetase . The cytosolic form of the enzyme was partially purified from rice cultured cells and assayed in the presence of millimolar concentrations of the compounds by means of three different assay methods, respectively measuring the hemibiosynthetic, the transferase, and the full biosynthetic reactions . Several compounds were found to exert a remarkable inhibition, with I(50) values similar to those obtained under the same conditions with a well-established inhibitor of glutamine synthetase, the herbicide phosphinothricin . Contrary to the reference compound, enzyme kinetics accounted for a reversible inhibition mechanism . The biological activity of the most active derivatives was further characterized by measuring free glutamine levels in cell suspension rice cultures following treatment with the inhibitors . Results confirmed their ability to interfere in vivo with nitrogen metabolism . A preliminary analysis of structure-activity relationship allowed it to be hypothesized that steric rather than electronic factors are responsible for the inhibitory potential of these compounds.

Anticancer Res, 2004 Mar-Apr, 24(2B), 827 - 31
Resistance developing after long-term ganciclovir prodrug treatment in a preclinical model of NSCLC; Kurdow R et al.; BACKGROUND: We recently demonstrated a 100% increase in the survival period with ganciclovir (GCV) therapy in mice hearing orthotopic HSV-TK-positive non-small cell lung cancer (NSCLC) tumors . However, long-term survival was not achieved . The aim of the present study was to evaluate tumor growth during extended GCV therapy and to monitor the herpes simplex virus thymidine kinase (HSV-TK) gene and protein in tumors at different time points . MATERIALS AND METHODS: The human NSCLC cell line KNS 62 was retrovirally transduced with the HSV-TK30 gene . Cell suspensions in which 100% or 25% of the cells were TK30-positive were inoculated subcutaneously in SCID bg mice . Tumor growth was evaluated during GCV therapy and HSV-TK DNA, RNA and protein were analyzed at different time points using PCR, RT-PCR and immunoblotting . RESULTS: HSV-TK DNA, RNA and TK30 protein were demonstrated in the tumors 21 days after subcutaneous tumor inoculation . TK-positive tumors regressed during GCV therapy and tumors in which 25% of the cells were TK-positive grew significantly more slowly than control tumors did . After 4 weeks of GCV therapy, HSV-TK DNA, RNA and TK protein were not detectable in the remaining tumors, which were therefore resistant to further GCV therapy . CONCLUSION: Prodrug therapy of the NSCLC cell line KNS 62, including bystander effects, is sufficient . Nevertheless, GCV-resistant tumors develop after functional loss of the TK gene . In the clinical context, further studies will need to evaluate immunological bystander effects or combinations with other drugs.

Leuk Lymphoma, 2004 Mar, 45(3), 529 - 38
Flow cytometry in the diagnosis of mediastinal tumors with emphasis on differentiating thymocytes from precursor T-lymphoblastic lymphoma/leukemia; Gorczyca W et al.; Flow cytometry (FC) has become the routine technique in the evaluation of hematopoietic neoplasms . Since the anterior mediastinum is a frequent site of involvement by both primary and secondary lymphoma/leukemia, flow cytometry plays an important role in the evaluation of mediastinal masses . The present study reviews 100 flow cytometry cases from patients presenting with mediastinal lesions . In 5 cases (5%) flow cytometry was not diagnostic due to either insufficient cell yield or low viability . In the remaining cases (95/100) cell suspensions were adequate for flow cytometry evaluation . Results showed that in 31/32 (96.8%), 2/3 (66.7%), 7/9 (77.8%), 7/8 (87.5%) and 11/11 (100%) cases of B-cell lymphoma, T-cell lymphoma, carcinoma, T-ALL/LBL and thymoma/thymic hyperplasia, respectively, the diagnosis could be reached by flow cytometry alone . Excluding HL, the general sensitivity of FC in diagnosing mediastinal tumors was approximately 92% . Among the 100 cases, flow cytometry gave non-informative results in 3 cases of diffuse large B-cell lymphoma, 1 case of peripheral T-cell lymphoma, and 3 cases of carcinoma . No false positive results were encountered . The phenotypic pattern, especially surface CD3 expression versus forward scatter, reliably discriminated between immature thymocytes from thymoma/thymic hyperplasia from T-ALL/LBL . Flow methodology has the advantage of rapid turn-around time as well as high sensitivity, enabling patients with large anterior mediastinal masses and/or superior vena cava syndrome to begin treatment as promptly as possible . In experienced hands, flow cytometry plays a valuable and complementary role to histology and immunohistochemistry in diagnosing mediastinal tumors.

Biochem Soc Trans, 2004 Jun, 32(Pt3), 524 - 8
Proteomic analysis of the Arabidopsis cell wall reveals unexpected proteins with new cellular locations; Slabas AR et al.; We initiated a proteomic study as part of a programme aimed at discovering novel functions of the plant cell wall . Cell-wall fragments isolated from cell-suspension cultures of Arabidopsis thaliana were stripped of protein sequentially using CaCl2 and a urea-based buffer . The protein fractions were separated by two-dimensional gel electrophoresis and individual proteins were identified by MS . We identified a number of proteins considered to be resident in other organelles but not the cell wall on the basis of their classical biological function . These included citrate synthase, which is known to be targeted to mitochondria, peroxisomes and glyoxysomes, and luminal binding protein, which is an ER (endoplasmic reticulum)-resident protein . Searches of the Arabidopsis database revealed that there are several genes encoding putative citrate synthase and luminal binding protein . We have also performed detailed analyses of the protein sequences and this paper shows how each one contains encrypted targeting information that results in the export of the protein to the extracellular matrix . We discuss the presence of alternative non-classical secretory pathways in plants .

Biol Reprod, 2004 Sep, 71(3), 942 - 7 Epub 2004 May 19.
Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture; Oatley JM et al.; Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny . The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals . The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF) . In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells . Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice . Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing . Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk . Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein . Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF . These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.

Chem Res Toxicol, 2004 May, 17(5), 623 - 32
Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro; Carvalho M et al.; Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported . The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated . Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes . The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h . alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD . The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase . No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h . In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes . The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities . The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites . The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.

Cent Eur J Public Health, 2004 Mar, 12 Suppl, S20 - 3
Evaluation of bronchoalveolar lavage fluid cytotoxic parameters after inhalation exposure to amosite and wollastonite fibrous dusts combined with cigarette smoke; Cerna S et al.; The aim of this work was to compare the influence of amosite-asbestos and wollastonite fibrous dusts combined with cigarette smoke on chosen cytotoxic parameters of bronchoalveolar lavage fluid (BALF) in rats . Fisher 344 rats inhaled wollastonite or amosite fibrous dusts (60 or 30 mg x m(-3) air) one hour every two days combined with daily breathing of diluted mainstream tobacco smoke (30 mg of TPM x m(-3) air) . The experiment lasted 6 months . After sacrifying the animals bronchoalveolar lavage (BAL) was performed and the viability and phagocytic activity of alveolar macrophages (AM), lactate dehydrogenase (LDH) and alkaline phosphatase activity (in the cell-free BALF), acid phosphatase (ACP) and cathepsin D activity (in cell-free BALF and BAL cell suspension) were examined . Exposure to amosite without tobacco smoke significantly decreased the viability of AM and increased the cathepsin D activity in BAL cells . Exposure to wollastonite significantly increased only the cathepsin D activity in BAL cells . Smoking significantly depressed the phagocytic activity of AM and amplified the amosite-induced increase of lysosomal enzyme activities--especially the activity of cathepsin D in BAL cells.

Cent Eur J Public Health, 2004 Mar, 12 Suppl, S11 - 3
Some lung cellular parameters reflecting inflammation after combined inhalation of amosite dust with cigarette smoke by rats; Beno M et al.; Cellular changes were followed in lung cell suspensions after 175 day inhalation by rats of concentrations 30 mg/m3 or 60 mg/m3 of amosite asbestos every second day combined with daily exposure to cigarette smoke at 30 mg of total particulate matter (TPM)/m3 air . Concomitantly, lung inflammation was assessed by changes in the bronchoalveolar lavage fluid (BALF) . A dose-dependent rise in the BALF inflammatory parameters was found . The rise of the proportion of binucleate (BNC) and multinucleate cells (MNC) in lung cell suspensions was also dose-dependent . It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of BNC and of MNC in lung cell suspensions may serve as a useful semiquantitative biomarker of the inflammation.

Pigment Cell Res, 2004 Jun, 17(3), 230 - 40
Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B; Hirobe T et al.; Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation . The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes . However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes . In this study, primary melanoblasts (c . 80%) and melanocytes (c . 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) . GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures . Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice . Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice . The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice . Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice . These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.

Eur Biophys J, 2004 Nov, 33(7), 589 - 95 Epub 2004 Nov.
Shear-induced alignment of self-associated hemoglobin in human erythrocytes: small angle neutron scattering studies; Garvey CJ et al.; Small angle neutron scattering (SANS) was performed on suspensions of actively metabolising human erythrocytes in the constant shear field induced by a Couette cell . The SANS pattern recorded on a two-dimensional detector was a function of the shear rate; at zero shear, the SANS pattern had radial symmetry around the direction of the beam . The radial average of the SANS pattern consisted of a broad intensity maximum superimposed on a decay . The intensity maximum at q = 0.1 A(-1) was attributed to isotropically oriented self-associated complexes of the tetrameric oxygen transport protein hemoglobin inside the erythrocytes . A flow curve of the cell suspension was used to identify at what shear rate a suspension of uniaxially oriented ellipsoidal cells is produced . The radial symmetry of the SANS patterns persisted until the shear rate was sufficient to produce a suspension of uniaxially oriented ellipsoidal cells . Again, an intensity maximum was present in directions parallel and orthogonal to the shear axis, but this intensity maximum was superimposed upon quite different intensity decays in each direction from that of the primary neutron beam . The angular range of the SANS instrument was limited, however the results from shear-induced structural changes is consistent with a model that involves hemoglobin complexes that are aligned with respect to the plasma membranes of the elongated cells.

Biotechnol Bioeng, 2004 Jun 20, 86(6), 612 - 21
Clearance of minute virus of mice by flocculation and microfiltration; Wickramasinghe SR et al.; Clearance of minute virus of mice (MVM) from CHO cell suspensions by flocculation and microfiltration has been investigated . MVM is a parvovirus that is recommended by the U.S . Food and Drug Administration for validating clearance of parvoviruses . The feed streams were flocculated using a cationic polyelectrolyte . Virus clearance in excess of 10,000-fold was obtained in the bulk permeate for flocculated feeds streams . However, the level of clearance was only about 10- to 100-fold for unflocculated feed streams . The results suggest that virus clearance involves interactions between the MVM particles, the cationic polyelectrolyte, and the CHO cells present . Validating virus clearance is a major concern in the biotechnology industry . New unit operations are frequently added to the purification train simply to validate virus clearance . However, many of these unit operations are less effective at validating clearance of nonenveloped viruses . Validating clearance of parvoviruses is often particularly problematic as they are nonenveloped and the virus particles are small (18 to 24 nm), making physical removal difficult . The results obtained herein indicate that addition of the cationic polyelectrolyte not only results in significant clearance of MVM but also leads to an increase in permeate flux .

Clin Cancer Res, 2004 May 1, 10(9), 3042 - 52
Intracellular patterns of Her-2/neu, ras, and ploidy abnormalities in primary human breast cancers predict postoperative clinical disease-free survival; Shackney SE et al.; PURPOSE: In an earlier study, the presence of aneuploidy, Her-2/neu overexpression, and ras overexpression in the same cells (triple-positive cells) was of prognostic significance (P < 0.015) in 91 patients with localized breast cancer (median follow up, 32 months) . Here, we present results involving a larger group of patients with longer follow-up . EXPERIMENTAL DESIGN: Fixed cell suspensions prepared from primary tumors of 189 patients with early breast cancer were studied prospectively by multiparameter flow cytometry . Correlated intracellular fluorescence-based measurements of cell DNA content and Her-2/neu and ras protein were obtained on each of >2000 cells in each tumor . Intracellular combinations of abnormalities in these measurements were correlated with subsequent patient disease-free survival (DFS) . Median time on study was 54 months (range, 7-128 months) . RESULTS: DFS of patients with > or = 5% triple-positive tumor cells was shorter than those who did not meet this criterion (P = 0.004) . The difference remained statistically significant after accounting for nodal status, tumor size, and each of the component abnormalities (P = 0.006) . Node-negative patients whose tumors had fewer than 2 abnormalities/cell had an especially favorable clinical course, with a 5-year DFS of 96% (lower confidence bound, 86%) . CONCLUSIONS: Patterns of accumulated intracellular molecular abnormalities in cells of primary human breast cancers are predictive for subsequent DFS independently of the abnormalities themselves taken individually.

Antioxid Redox Signal, 2004 Jun, 6(3), 677 - 86
Using EPR to measure a critical but often unmeasured component of oxidative damage: oxygen; Swartz HM; Oxygen is a critical variable in oxidative damage . It can be a direct reactant in one or more of the pertinent reactions that result in oxidative damage . It also is an essential substrate for mitochondrial respiration and many other essential synthetic and degradative reactions . The level of oxygen can have a regulatory role, affecting the rate and direction of metabolic processes and physiological functions that are germane to the pathophysiological processes that are being studied . Its supply to tissue and to cells is therefore a critical parameter governing normal homeostasis . The level of oxygen at specific sites may affect cell signaling . It therefore seems clear that it can be very useful to measure oxygen when studying oxidative damage . In order for the measurements of oxygen to be most useful, it often is essential to measure the amount of oxygen at particular sites and under appropriate conditions . These needs require methodology that can make sensitive and localized measurements of oxygen . Electron paramagnetic resonance (EPR) oximetry is such a technique, plus it has the capability of making repeated measurements from the same site non-invasively . The principles and applications of EPR oximetry to viable systems, including cell suspensions and intact animals, are described in this paper.

J Plant Physiol, 2004 Apr, 161(4), 355 - 61
Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures; Qin WM et al.; After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied . The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells . The H2O2 forming enzyme was also investigated . The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor . Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter . The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.

J Bacteriol, 2004 May, 186(10), 3022 - 8
Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens; Coppi MV et al.; Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones . An examination of the G . sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases . In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement . Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors . Hya, in contrast, was not . These findings suggest that Hyb is an essential respiratory hydrogenase in G . sulfurreducens.

J Surg Res, 2004 Jun 1, 119(1), 29 - 35
Direct visualization of nitric oxide release by liver cells after the arrest of metastatic tumor cells in the hepatic microvasculature; Qi K et al.; BACKGROUND: Our previous studies have shown that the injection of B16F1 melanoma cells into the mesenteric vein can induce the rapid local release of nitric oxide (NO) in the liver, causing apoptosis of the melanoma cells in the liver sinusoids and inhibiting the subsequent formation of hepatic metastases . In this study, we have investigated the distribution and cellular source of NO in this model . MATERIALS AND METHODS: In situ liver perfusion was established in both wild-type (wt) and endothelial nitric oxide synthase knockout (eNOS KO) C57BL/6 mice . A specific fluorescent NO probe, 4,5-diaminofluorescein diacetate (DAF-2 DA) (5 micromol/L), was perfused into the portal venous system to label the liver tissue . Then, a MitoTracker Orange labeled B16F1 melanoma cell suspension (2 x 10(6) cells/ml) was injected through a portal vein catheter by a peristaltic pump . Images of the liver tissue were taken by confocal microscopy from a selected area to determine the cellular source of NO . For quantification, the fluorescence intensity of this area was measured over time by Fluoview software . RESULTS: Diaminotriazolofluorescein (DAF-2T) fluorescence (indicating NO generation) was detected in hepatic parenchymal cells located in the periportal region in both wt C57BL/6 and eNOS KO C57BL/6 mice and was intensified by increased flow rate in the portal venous system . The B16F1 cells arrested in the periportal sinusoids, corresponding to zone 1 of the hepatic acinus . DAF-2T fluorescence was expressed by both sinusoidal lining cells and hepatocytes at the site of tumor cell arrest . The fluorescence intensity of these cells increased approximately 2-fold over a time of 500 s . In contrast, there was no increase in the fluorescence intensity of the sinusoidal lining cells and hepatocytes in mice perfused with buffer or in eNOS KO mice perfused with B16F1 cells . CONCLUSION: This study demonstrates that NO is produced by hepatic parenchymal cells mainly located in the periportal zones and that the arrest of the B16F1 melanoma cells causes an eNOS-dependent local burst of NO by the sinusoidal lining cells and hepatocytes in the periportal areas.

Pharmazie, 2004 Apr, 59(4), 323 - 4
Identification and determination of invertase secreted by tomato cells; Stano J et al.; A simple and rapid procedure for the identification and determination of extracellular invertase from a culture medium of tomato cell suspension cultures is described . Sucrose was used as substrate for the determination of the extracellular and intracellular activities of the enzyme . The culture medium (without cells) was used for identification and determination of extracellular enzyme activity . Intracellular activity was estimated from the cell suspension.

Comp Biochem Physiol A Mol Integr Physiol, 2004 Mar, 137(3), 479 - 93
Lobster hepatopancreatic epithelial single cell suspensions as models for electrogenic sodium-proton exchange; Mandal PK; Sodium-proton antiporters, also called Na+/H+ exchangers (NHE), are vital transmembrane proteins involved in multiple cellular functions including transepithelial ion transport and Na+ homeostasis of cells throughout the biological kingdom . Na+/H+ exchange is accelerated by cytosolic acidification and also by osmotically induced cell shrinking, thereby promoting recovery of the physiological pHi and volume . Eight isoforms of Na+/H+ exchangers have been cloned and characterized to date and share the same overall structure, but exhibit differences with respect to cellular localization, kinetic variables and plasma membrane targeting, in polarized epithelial cells . The electrogenic Na+ absorption across tight epithelia from invertebrates follow significantly different principles from the electroneutral Na+/H+ antiporter found in vertebrates . In all invertebrate cells examined, the antiporter displayed a 2Na+/1H+ transport stoichiometry and this transport was markedly inhibited by exogenous calcium and zinc . Na+/H+ exchangers (NHE) are present in crustacean hepatopancreatic cell type suspensions and are believed to function in acid-base regulation by driving the extrusion of protons across the hepatopancreatic epithelium in exchange for Na+ in the sea water . A brief review of current knowledge about Na+/H+ exchangers has been presented . In addition, understanding of hepatopancreatic Na+/H+ exchange is described as obtained after isolation of purified E-, R-, F- and B-cell suspensions from the whole organ by centrifugal elutriation.

Plant Physiol, 2004 May, 135(1), 516 - 29 Epub 2004 Apr 30.
Analysis of nitric oxide signaling functions in tobacco cells challenged by the elicitor cryptogein; Lamotte O et al.; Nitric oxide (NO) has recently emerged as an important cellular mediator in plant defense responses . However, elucidation of the biochemical mechanisms by which NO participates in this signaling pathway is still in its infancy . We previously demonstrated that cryptogein, an elicitor of tobacco defense responses, triggers a NO burst within minutes in epidermal sections from tobacco leaves (Nicotiana tabacum cv Xanthi) . Here, we investigate the signaling events that mediate NO production, and analyze NO signaling activities in the cryptogein transduction pathway . Using flow cytometry and spectrofluorometry, we observed that cryptogein-induced NO production in tobacco cell suspensions is sensitive to nitric oxide synthase inhibitors and may be catalyzed by variant P, a recently identified pathogen-inducible plant nitric oxide synthase . NO synthesis is tightly regulated by a signaling cascade involving Ca2+ influx and phosphorylation events . Using tobacco cells constitutively expressing the Ca2+ reporter apoaequorin in the cytosol, we have shown that NO participates in the cryptogein-mediated elevation of cytosolic free Ca2+ through the mobilization of Ca2+ from intracellular stores . The NO donor diethylamine NONOate promoted an increase in cytosolic free Ca2+ concentration, which was sensitive to intracellular Ca2+ channel inhibitors . Moreover, NO appears to be involved in the pathway(s) leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethylene-forming enzyme cEFE-26, and cell death . In contrast, NO does not act upstream of the elicitor-induced activation of mitogen-activated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes . Collectively, our data indicate that NO is intimately involved in the signal transduction processes leading to cryptogein-induced defense responses.

Plant Physiol Biochem, 2004 Apr, 42(4), 299 - 306
Sucrose synthase isoforms in cultured tobacco cells; Matic S et al.; The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose (UDP-Glc) and fructose . The enzyme exists in different isoforms and is both located in the cytosol, membrane-bound and associated to the actin cytoskeleton . We here investigate sucrose synthase from tobacco (Nicotiana tabacum L.) BY-2 heterotrophic cell suspensions . Two different isoforms of sucrose synthase SuSy1 and SuSy2, could be purified from cytosolic extracts of these cells using a combination of poly(ethylene glycol) (PEG) precipitation, gel filtration, ion-exchange chromatography and affinity chromatography . They were clearly distinct, both with regard to the binding to the ion-exchange column and with regard to their kinetic and regulatory properties . SuSy1, the more abundant species, showed lower V(max) and K(m) for sucrose and UDP compared to the less abundant SuSy2 . The activity of SuSy2 in the breakdown direction was stimulated by 60% by actin, in contrast to that of SuSy1, which showed a 17% inhibition . An indication of interaction between SuSy1 and actin was obtained by partitioning in aqueous Dextran-PEG two-phase systems . Furthermore, fructose 2,6-bisphosphate (F26BP) at micromolar concentrations stimulated SuSy2 in the presence of actin while SuSy1 was strongly inhibited by fructose . Possible roles of these two isoforms in the sucrose turnover in BY-2 cells are discussed.

Biochim Biophys Acta, 2004 May 3, 1691(2-3), 79 - 90
Effects of Li+ transport and intracellular binding on Li+/Mg2+ competition in bovine chromaffin cells; Fonseca CP et al.; Li(+) transport, intracellular immobilisation and Li(+)/Mg(2+) competition were studied in Li(+)-loaded bovine chromaffin cells . Li(+) influx rate constants, k(i), obtained by atomic absorption (AA) spectrophotometry, in control (without and with ouabain) and depolarising (without and with nitrendipine) conditions, showed that L-type voltage-sensitive Ca(2+) channels have an important role in Li(+) uptake under depolarising conditions . The Li(+) influx apparent rate constant, k(iapp), determined under control conditions by (7)Li NMR spectroscopy with the cells immobilised and perfused, was much lower than the AA-determined value for the cells in suspension . Loading of cell suspensions with 15 mmol l(-1) LiCl led, within 90 min, to a AA-measured total intracellular Li(+) concentration, {Li(+)}(iT)=11.39+/-0.56 mmol (l cells)(-1), very close to the steady state value . The intracellular Li(+) T(1)/T(2) ratio of (7)Li NMR relaxation times of the Li(+)-loaded cells reflected a high degree of Li(+) immobilisation in bovine chromaffin cells, similar to neuroblastoma, but larger than for lymphoblastoma and erythrocyte cells . A 52% increase in the intracellular free Mg(2+) concentration, Delta{Mg(2+)}(f)=0.27+/-0.05 mmol (l cells)(-1) was measured for chromaffin cells loaded with the Mg(2+)-specific fluorescent probe furaptra, after 90-min loading with 15 mmol l(-1) LiCl, using fluorescence spectroscopy, indicating significant displacement of Mg(2+) by Li(+) from its intracellular binding sites . Comparison with other cell types showed that the extent of intracellular Li(+)/Mg(2+) competition at the same Li(+) loading level depends on intracellular Li(+) transport and immobilisation in a cell-specific manner, being maximal for neuroblastoma cells.

Bioorg Med Chem, 2004 May 15, 12(10), 2781 - 6
Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis; Gerasimenko I et al.; The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta . The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine . An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration . Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity . SDS-PAGE analysis showed a Mr for the enzyme of approximately 50 kDa . The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced . The enzyme preparation (> 875-fold enrichment) delivering the N-terminal sequence was isolated from R . serpentina cell suspensions . Sequence alignment of the five peptides showed highest homologies in a range of 30-71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis . Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.

Cytometry B Clin Cytom, 2004 May, 59B(1), 10 - 23
A suitable method for identifying cell aggregates in laser scanning cytometry listmode data for analyzing disaggregated cell suspensions obtained from human cancers; Shackney SE et al.; BACKGROUND: The presence of cell aggregates in cell suspensions obtained from human solid tumors can interfere with the measurement of cell DNA content of cell singlets, and can confound multiparameter analysis of other measurements on the same cells . Flow cytometric corrections for cell aggregates based on signal pulse shape have not proven to be reliable . Mathematical models have been developed to correct for cell aggregates in binned DNA histogram data, but they are not suitable for the correction of correlated non-DNA measurements obtained on the same cells . METHODS: A total of 21 samples representing a variety of normal and malignant human cell types, including normal lymphocytes, normal sputum, human breast cancer cell lines, and mechanically disaggregated cell suspensions from primary breast cancers and nonsmall cell lung cancers, were studied by laser scanning cytometry (LSC) using the CompuCyte laser scanning cytometer (Cambridge, MA) . Nuclear area, nuclear perimeter, and an LSC-based cell texture parameter were measured on approximately 400 cells in each sample, using an air-cooled, violet laser emitting at a wavelength of 405 nm for DAPI excitation, and each cell was classified as a singlet or aggregate by its appearance under direct observation . A "saddle function" provided by CompuCyte was used, together with an algorithm based on the measurements of nuclear area, perimeter, and cell texture (the APT algorithm), to identify cell aggregates and exclude them from the listmode data file . RESULTS: Proportions of cell aggregates in the uncorrected samples ranging from 6 to 56% (mean, 20%) were reduced to proportions ranging from 0 to 7% (mean, 2.4%) after correction . The discriminant function was "tuned" to maintain both average cell singlet purity and average cell singlet yield at >70% over a broad range of cell DNA contents . CONCLUSIONS: A combined approach to cell aggregate detection, which utilizes both the saddle function and the APT algorithm, produces list mode data files that exclude >80% of cell aggregates from samples of disaggregated cell suspensions of human tumors and other sources of clinical material . Such data files are suitable for multiparameter analysis .

Methods Mol Biol, 2004, 282, 85 - 101
Immunoassay for single-stranded DNA in apoptotic cells; Frankfurt OS; The apoptosis assay described in this chapter is based on the selective denaturation of DNA in condensed chromatin of apoptotic cells and the detection of denatured DNA with a monoclonal antibody highly specific to single-stranded DNA . Optimal results are obtained by the heating at a relatively low temperature in the presence of formamide . The assay detects apoptotic cells but not necrotic cells or cells with DNA breaks in the absence of apoptosis . The sensitivity of the assay reflects the detection of early and late apoptosis . Apoptotic cells are detected in the sections of frozen or formalin-fixed paraffin-embedded tissues by immunohistochemistry and in the cell suspensions by flow cytometry or fluorescence microscopy . Apoptosis enzyme-linked immunoassay based on DNA denaturation by formamide in microtiter plates and one-step immunostaining is applied for high-throughput screening of drugs . The enzyme-linked immunoassay has the ability to distinguish anticancer drugs from toxic chemicals, to predict selective toxicity to cancer cells, and to detect drug synergism.

Int Immunopharmacol, 2004 Apr, 4(4), 563 - 9
Immunomodulation and antitumor activity by a polysaccharide-protein complex from Lycium barbarum; Gan L et al.; The modulation of a polysaccharide-protein complex from Lycium barbarum (LBP3p) on the immune system in S180-bearing mice was investigated . The mice inoculated with S180 cell suspension were treated p.o . with LBP3p (5, 10 and 20 mg/kg) for 10 days . The effects of LBP3p on transplantable tumors and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells (QHS), lymphocyte proliferation, the activity of cytotoxic T lymphocyte (CTL), interleukin-2 (IL-2) gene expression and lipid peroxidation were studied . LBP3p could significantly inhibit the growth of transplantable sarcoma S180 and increase macrophage phagocytosis, the form of antibody secreted by spleen cells, spleen lymphocyte proliferation, CTL activity, IL-2 mRNA expression level and reduce the lipid peroxidation in S180-bearing mice . The effect is not dose-dependent in a linear fashion . A total of 10 mg/kg dose is more effective than 5 and 20 mg/kg doses . This suggests that LBP3p at 10 mg/kg has a highly significant effect on tumor weight and improves the immune system .

Acta Biochim Pol, 2004, 51(1), 219 - 22
Cadmium-induced changes in antioxidant enzymes in suspension culture of soybean cells; Sobkowiak R et al.; Cadmium (Cd), similarly to other heavy metals, inhibits plant growth . We have recently showed that Cd(2+) either stimulates (1-4 microM) or inhibits (>/= 6 microM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak & Deckert, 2003, Plant Physiol Biochem . 41: 767-72) . Here, soybean cell suspension cultures were treated with various concentrations of Cd(2+) (1-10 microM) and the following enzymes were analyzed by native electrophoresis: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APOX) . We found a significant correlation between the cadmium-induced changes of soybean cell culture growth and the isoenzyme pattern of the antioxidant enzymes . The results suggest that inhibition of growth and modification of antioxidant defense reactions appear in soybean cells when Cd(2+) concentration in culture medium increases only slightly, from 4 to 6 microM.

Exp Cell Res, 2004 May 1, 295(2), 539 - 48
Human immunodeficiency virus type 1 Tat protein modulates cell cycle and apoptosis in Epstein-Barr virus-immortalized B cells; Colombrino E et al.; Patients infected with human immunodeficiency virus type 1 (HIV-1) develop a spectrum of B cell lymphoproliferative disorders ranging from polyclonal B cell activation to B cell lymphomas . While a direct role of Epstein-Barr virus (EBV) is well recognized for most of these lesions, recent findings have suggested that transactivator HIV-1 Tat protein might be involved in the pathogenesis of B cell lymphomas . Tat-expressing EBV-positive B cells were generated by transduction with a retroviral Tat-encoding vector . B(Tat+) cells expressed lower levels of anti-apoptotic protein Bcl-2 than parental and control B(Tat-) cells, generated by transduction with an empty retroviral vector, and were more prone to apoptosis upon serum withdrawal, as assessed by analysis of annexin V-stained cells and cleavage of poly-ADP-ribose-polymerase by caspase 3 . Nevertheless, in serum starvation, B(Tat-) cells mainly exhibited the Rb hypo-phosphorylated form, underwent cell cycle arrest, and grew in single cell suspension, while B(Tat+) cells displayed the Rb hyper-phoshorylated form, progressed throughout the cell cycle, and retained the ability to grow in small clumps . Finding that B(Tat+) cells maintained proliferative capacity upon serum withdrawal suggests that cells expressing Tat have growth advantages among the EBV-driven cell proliferations and may originate B cell clones with more oncogenic potential.

Biorheology, 2004, 41(2), 113 - 25
Platelet near-wall excess in porcine whole blood in artery-sized tubes under steady and pulsatile flow conditions; Xu C et al.; Platelet margination (enhanced platelet concentration in the near wall region of a blood vessel) has been well documented in small vessels . In artery-sized vessels margination has only been demonstrated in one study, using ghost cell suspensions and under relatively non-physiologic conditions of steady flow and 50 cm development length . Local sampling experiments were performed to confirm platelet margination in artery-sized stainless steel tubes, for a typical anatomical length and under pulsatile flow, using fresh EDTA-anticoagulated porcine whole blood (N=21) . Experiments were designed using three-dimensional Computational Fluid Dynamics (CFD) to model the sample region with greater fidelity . Steady flow experiments in 50 cm long tubes verify published laser Doppler measurements of platelet margination in 3 mm ID tubes at normal arterial shear rate (500 s(-1) . Margination persists under pulsatile flow conditions (63.8 pulses/min), but in steady flow at length of 10 cm, margination is reduced . Platelet margination ratio (the ratio of the platelet concentration near the wall to bulk average platelet count) ranges from 1.21 to 2.48 . No significant effects of calculated sampling thickness (20 microm and 50 microm) or pulsatility were detected . Hematocrit margination ratio is 0.68 to 0.90 . Two model platelet concentration profiles are fit to the experimental results.

Kidney Int, 2004 May, 65(5), 1604 - 14
Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney; Iwatani H et al.; BACKGROUND: Although the regenerative stem cell is expected to exist in many adult tissues, the cell contributing to the regeneration of the kidney remains unknown in its type and origin . METHODS: In this study, we isolated cells that show low stain with a DNA-binding dye Hoechst 33342 (Hoechst(low) cells) from adult rat kidney, and investigated their differentiation potentials . RESULTS: Hoechst(low) cells, generally termed side population cells, existed at a frequency of 0.03% to 0.1% in the cell suspension of the digested kidney . Analysis of the kidney-derived Hoechst(low) cells after bone marrow transplantation indicated that some of the cells were derived from bone marrow . When enhanced green fluorescent protein (EGFP)-labeled kidney-derived Hoechst(low) cells were intravenously transplanted into wild-type adult rats, EGFP(+) cells were not detected in the kidney, but EGFP(+) skeletal muscle, EGFP(+) hepatocytes and EGFP(+) bone marrow cells were observed . Even after the induction of the experimental glomerulonephritis and gentamicin-induced nephropathy that promote the differentiation of bone marrow-derived cells into repopulating mesangial cells and tubular component cells, respectively, EGFP(+) mesangial or tubular cells were not observed . Neither with an in vitro system, which we established to produce mesangial-like cells from crude bone marrow culture, did Hoechst(low) cells yield mesangial-like cells . CONCLUSION: These findings implicate that Hoechst(low) cells in the kidney may have potentials for hematopoietic and nonhematopoietic lineages, but are not stem cells for renal cells, especially mesangial and tubular cells.

Plant J, 2004 May, 38(3), 410 - 20
A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings; Perruc E et al.; A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A . thaliana cell suspension cultures challenged with osmotic stress . The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only . Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus . Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor . AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity . Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth . By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type . Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.

J Invest Dermatol, 2004 Mar, 122(3), 670 - 2
Ontogeny of Langerin/CD207 expression in the epidermis of mice; Tripp CH et al.; C-type lectin receptors help Langerhans cells (LC) to take up and process pathogens . Langerin/CD207 is a mannose-binding C-type lectin that is specifically expressed by LC . It is involved in antigen uptake in an as yet poorly defined way, and it is a major molecular constituent of Birbeck granules . We studied the emergence of Langerin expression in LC in epidermal sheets and cell suspensions during ontogeny . Langerin appears later than MHC II expression . Intracellular Langerin expression becomes apparent 2-3 d after birth . Only 10 days after birth all LC co-express Langerin . The intensity of Langerin expression reaches adult levels by 3 wk after birth . Early Langerin expression appears to correlate at least in part with the physical presence of Birbeck granules.

Andrologia, 2004 Apr, 36(2), 69 - 77
Complex nature of the human antisperm antibody response in SCID mice; Kurpisz M et al.; Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely-combined immunodeficient (SCID) mice in concentrations of 2.5-4.0 x 10(7) cells per mouse . Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa . In some experiments, immunodepletion of CD8(+) cells was carried out prior to grafting . Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens) . An enzyme-linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated . We have been successful by generating primary and secondary immune responses with 'naive' human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8(+) immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice . We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from 'naive' or pre-sensitized in vivo subjects . It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre-primed to spermatozoa) individuals . A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.

Exp Neurol, 2004 May, 187(1), 58 - 64
An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality; Sortwell CE et al.; Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease . Survival rates of grafted dopamine (DA) neurons are extremely poor (5-20%) and is even poorer in grafts to the aged striatum . Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates . However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions . This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting . Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days . All grafts were placed in the denervated striatum of young adult male Fischer 344 rats . Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts . Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery . Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days . Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts . This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.

Thromb Res, 2004, 113(1), 75 - 83
The effect of TNF-alpha, PMA, and LPS on plasma and cell-associated IL-8 in human leukocytes; Lund T et al.; INTRODUCTION/AIM: This study was performed to examine the proficiency of mononuclear cells (MNC) and polymorphonuclear cells (PMN) in a whole blood model to expressing interleukin-8 (IL-8) in response to various stimuli . METHODS: Isolated cells that had been recombined with heparinized plasma were incubated with lipopolysaccharide (LPS), phorbol myristate acetate (PMA) and tumor necrosis factor (TNF)-alpha . RESULTS: IL-8 release by MNC was most potently induced by LPS, reaching significant levels after 2-h incubation in the presence of 0.2 ng/ml LPS . In contrast, 5.0 ng/ml LPS was required for PMN to release significant amounts of the cytokine (P<0.001) . When PMN and MNC were coincubated (MNC/PMN), LPS-induced IL-8 release was reduced compared to the release from MNC alone, regardless of the concentration of LPS used . IL-8 release by PMN was much more strongly induced by TNF-alpha, increasing by 1050% in the presence of 10 ng/ml TNF-alpha (P<0.005), whereas MNC or MNC/PMN subjected to this stimulus alone did not significantly enhance their IL-8 release . PMA had no effect on IL-8 release from either cell type . Since a high portion of IL-8 in blood is associated with cells, the IL-8 levels in isolated and lysed cell suspensions were also quantified . Thus, a considerably higher level of IL-8 was found in freshly isolated PMN (0.58+/-0.09 ng/ml) than in MNC (0.010+/-0.007 ng/ml) . PMN remained the main source for cell-associated IL-8 after 2-h incubation in the absence of any added stimuli, harbouring a relatively high level of the cytokine (3.37+/-1.38 ng/ml), which was significantly enhanced in the presence of TNF-alpha (8.99+/-1.46 ng/ml, P<0.001) . CONCLUSION: This study shows that LPS is an effective inducer of IL-8 in MNC, whereas TNF-alpha is a potent agonist for IL-8 release from PMN . The main portion of cell-associated IL-8 is present in PMN when the cells are stimulated in their normal environment of plasma.

Clin Cancer Res, 2004 Apr 1, 10(7), 2512 - 24
Treatment of HER-2/neu overexpressing breast cancer xenograft models with trastuzumab (Herceptin) and gefitinib (ZD1839): drug combination effects on tumor growth, HER-2/neu and epidermal growth factor receptor expression, and viable hypoxic cell fraction; Warburton C et al.; PURPOSE: The purpose of this research was to assess the effects of single agent and combination treatment with trastuzumab and gefitinib on tumor growth and tumor microenvironment in two HER-2/neu overexpressing breast xenograft models, MDA-MB-435/LCC6(HER-2) (LCC6(HER-2); estrogen receptor negative) and MCF-7(HER-2) (estrogen receptor positive) . EXPERIMENTAL DESIGN: LCC6(HER-2) and MCF-7(HER-2) cells, both in tissue culture and xenografts grown in SCID-Rag 2M mice, were treated with trastuzumab and gefitinib, alone or in combination . The rate of tumor growth was determined . In addition, tumor HER-2/neu and epidermal growth factor receptor expression, cell viability, cell cycle distribution, and proportion of viable hypoxic cells were determined by flow cytometric analyses of single tumor cell suspensions . RESULTS: Both tumor models were very sensitive to trastuzumab and moderately sensitive to gefitinib in vivo . The combination resulted in therapeutic effects, as judged by inhibition of tumor growth, which was greater (albeit not statistically significant) than that observed with trastuzumab administered as a single agent . Trastuzumab was effective in down-regulating HER-2/neu, and gefitinib mediated a reduction in epidermal growth factor receptor expression on tumor cells . In LCC6(HER-2) tumors, trastuzumab significantly reduced tumor cell viability, which was not improved by the addition of gefitinib . Gefitinib dramatically reduced the proportion of viable hypoxic cells in LCC6(HER-2) and MCF-7(HER-2) tumors . This effect was abrogated by the addition of trastuzumab . CONCLUSIONS: Although in vivo efficacy studies in two HER-2/neu overexpressing breast xenograft models showed that the combination of trastuzumab and gefitinib was effective, analyses of various cellular parameters failed to reveal beneficial effects and argue that this drug combination may not be favorable.

Int J Artif Organs, 2004 Feb, 27(2), 127 - 36
Fibrin/Schwann cell matrix in poly-epsilon-caprolactone conduits enhances guided nerve regeneration; Galla TJ et al.; The goal of this study was to investigate if a three dimensional matrix, loaded homogeneously with Schwann cells and the neurotrophic factor LIF (leukemia inhibitory factor), enhances regeneration in a biodegradable nerve guidance channel as compared to non-structured cell suspensions . Therefore a 10 mm nerve gap in the buccal branch of the rat's facial nerve was bridged with tubular PCL (poly-epsilon-caprolactone) conduits filled with no matrix, Schwann cells, the three dimensional fibrin/Schwann cell matrix or the fibrin/Schwann cell matrix added with LIF Four weeks after the nerve defects were bridged histological and morphometric analyses of the implants were performed . In conclusion, the three dimensional fibrin/Schwann cells matrix enhanced the quantity and the quality of peripheral nerve regeneration through PCL conduits . The application of LIF prevented hyperneurotization . Therefore, tissue engineered fibrin/Schwann cells matrices are new invented biocompatible and biodegradable devices for enhancing peripheral nerve regeneration as compared to non-structured cell suspensions without neurotrophic factors.

Plant Cell Rep, 2004 Jul, 22(12), 951 - 8 Epub 2004 Apr 06.
Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies; Chong TM et al.; The effects of medium strategies {maintenance (M), intermediary (G), and production (P) medium} on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated . These were compared with third-stage leaf and 1-month-old callus culture . With P medium strategy, cell growth at 49 g l(-1), intracellular AQ content at 42 mg g(-1) DW, and H2O2 level at 9 micromol g(-1) FW medium were the highest as compared to the others . However, the extent of lipid peroxidation at 40.4 nmol g(-1) FW and total carotenoids at 13.3 mg g(-1) FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels . Vitamin C content at 30-120 microg g(-1) FW in all culture systems was almost half the leaf content . On the other hand, vitamin E content was around 400-500 microg g(-1) FW in 7-day-old cultures from all medium strategies and reduced to 50-150 microg g(-1) FW on day 14 and 21; as compared to 60 microg g(-1) FW in callus and 200 microg g(-1) FW in the leaf . This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures . When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2004 Mar, 18(2), 123 - 6
{Preliminary study on research method of cell survival rate at procedure of cryopreservation of tissue engineered tendons}; Zhu XQ et al.; OBJECTIVE: To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons . METHODS: In the 4th generation of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342 (Ho) . The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguish them . The seeding cells were collected to make them to be the cell suspension of suitable concentration (6.0 x 10(5) cell/ml) before they were divided into two parts . We cryopreserved and defrosted one part three times to kill the cells and did not cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to non-cryopreserved cells . The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreserved cell to non-cryopreserved cell and the cell survival rates . We compared the cell survival rates between immediate flow cytometry and that 2 hours after fluorescence staining . RESULTS: The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r = 0.970, P < 0.05), image analysis study also showed the correlation was significant (r = 0.982, P < 0.05) . The cell survival rate decreased by use of flow cytometry two hours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (P > 0.05) . We could also observe the cells on the tissue engineered tendons by fluorescence image directly . CONCLUSION: Flow cytometry and fluorescence image after PI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservation of tissue engineered tendons.

Planta, 2004 Jul, 219(3), 428 - 39 Epub 2004 Apr 03.
Photoinhibition and recovery in a herbicide-resistant mutant from Glycine max (L.) Merr . cell cultures deficient in fatty acid unsaturation; Alfonso M et al.; Photoinhibition and recovery were studied in two photosynthetic cell suspensions from soybean (Glycine max L . Merr): the wild type (WT) and the herbicide-resistant D1 mutant STR7 . This mutant also showed an increase in saturated fatty acids from thylakoid lipids . STR7 was more sensitive to photoinhibition under culture conditions . In vivo photoinhibition experiments in the presence of chloramphenicol, in vitro studies in isolated thylakoid membranes, and immunoblot analysis indicated that the process of light-induced degradation of the D1 protein was not involved in the response of STR7 to light . At growth temperature (24 degrees C), the recovery rate of photoinhibited photosystem II (PSII) was slower in STR7 relative to WT . Photoinhibition and recovery were differentially affected by temperature in both cell lines . The rates of photoinhibition were faster in STR7 at any temperature below 27 degrees C . The rates of PSII recovery from STR7 were more severely affected than those of WT at temperatures lower than 24 degrees C . The photoinhibition and recovery rates of WT at 17 degrees C mimicked those of STR7 at 24 degrees C . In organelle translation studies indicated that synthesis and elongation of D1 were substantially similar in both cell lines . However, sucrose gradient fractionation of chloroplast membranes demonstrated that D1 and also other PSII proteins such as D2, OEE33, and LCHII had a reduced capability to incorporate into PSII to yield a mature assembled complex in STR7 . This effect may become the rate-limiting step during the recovery of photoinhibited PSII and may explain the increased sensitivity to high light found in STR7 . Our data may hint at a possible role of fatty acids from membrane lipids in the assembly and dynamics of PSII.

Ann Hematol, 2004 May, 83(5), 307 - 8 Epub 2003 Oct 14.
Delayed hemolytic transfusion reaction due to anti-S antibody in patient with anti-Jk(a) autoantibody and multiple alloantibodies; Guastafierro S et al.; We describe the case of a 60-year-old woman with a delayed hemolytic transfusion reaction (DHTR) . She had a history of an ulcerative colitis, blood transfusion because of rectal bleeding, and surgical removal of descendent and sigmoid colon . At admission, laboratory data showed Hb 6.3 g/dL, reticulocytes 120 x 10(9)/L, serum total bilirubin 1.2 mg/dL (direct bilirubin: 0.2 mg/dL) . Pretransfusion antibody screening procedures were positive . A monospecific autoanti-Jk(a) and three alloantibodies (anti-c, -E, -K) were identified by immunohematologic studies . The patient received two units of crossmatch compatible concentrated red blood cells . Six days later biochemical serum values showed Hb 6.2 g/dL, LDH 975 I.U./L and total bilirubin 2.95 mg/dL (direct 0.35 mg/dL) . Crossmatches with red cell suspension of transfused blood units and a post-transfusion serum were repeatedly positive . Laboratory tests showed the presence of anti-S alloantobody in the serum and eluate . Moreover, pre-transfusion serum of the patient was retrospectively retested: anti-S was not detected . These data suggested a DHTR . The present case is unusual and interesting because of the association of a rare autoanti-Jk(a), non responsible for anemia, and four alloantibodies of which anti-S involved in a DHTR.

Plant Physiol, 2004 Apr, 134(4), 1414 - 26 Epub 2004 Apr 02.
Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants; Rea G et al.; To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes . Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines . Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified . Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates . These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants . The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity . The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Mol Cell Proteomics, 2004 Jul, 3(7), 675 - 91 Epub 2004 Apr 01.
Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome; Marmagne A et al.; Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism . Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented . Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported . To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins . We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests . Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies . The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments . The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy . An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism . This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Eur J Gynaecol Oncol, 2004, 25(1), 27 - 32
Differential expression of CD40 and CD95 in ovarian carcinoma; Ciaravino G et al.; PURPOSE: The role of CD95 (Fas) as a mediator of apoptosis has been well documented . CD40 ligation has been recently shown to initiate apoptosis and modulate CD95 mediated apoptosis in normal and some neoplastic tissues . Here we report the expression of CD95 and CD40 in cryopreserved cell suspensions from ovarian cancer associated ascites, fresh primary and recurrent ovarian carcinoma (OVCA) specimens, and ten established ovarian cancer cell lines . The effect of CD95 and CD40 receptor binding on apoptosis is described in two cell lines . EXPERIMENTAL DESIGN: Ascites specimens, fresh primary and recurrent OVCA specimens were dissociated to single cell suspensions . Expression of CD95 and CD40 was analyzed using flow cytometry . Apoptosis was determined via annexin uptake by flow cytometry following incubation with anti-CD95 antibody, CH11 and trimeric CD40L . RESULTS: Ascites showed the highest expression of both CD95 and CD40 . Recurrent OVCA, in contrast, expressed low levels of CD95 and CD40 . Primary OVCA showed moderate expression of both receptors . CD40 expression in ascites was significantly greater when compared to solid specimens (p < 0.05) . Both CD40 and CD95 were strongly expressed in eight of ten cell lines studied . Binding of CD40L did not influence CD95 mediated apoptosis . CONCLUSIONS: CD40 is ubiquitously expressed in ovarian carcinomas and expression differs between ascites and solid tumor . There may be differential expression of both CD40 and CD95 in recurrent vs primary ovarian carcinoma, which may contribute to increased clinical malignancy of recurrent disease . In contrast to other epithelial malignancies, CD40 ligation does not appear to modulate CD95 mediated apoptosis.

Toxicol In Vitro, 2004 Jun, 18(3), 359 - 64
Determination of sunscreen immune protection factors using human dendritic cell suspensions; Peguet-Navarro J et al.; In a previous study, we have used UVB-irradiated human skin explants and the allostimulatory function of Langerhans cells (LC) to determine immune protection factors (IPF) for sunscreens . We sought here to simplify the model by using either human enriched LC suspensions or in vitro generated dendritic cells from human monocytes (MoDC) . LC or MoDC suspensions were irradiated with increasing doses of UVB through a piece of translucent strip recovered or not with the sunscreens . The allostimulatory function of the cells was then analysed in a mixed lymphocyte reaction and the UVB dose providing 50% immunosuppression (D50%) was determined graphically . IPF were determined by the ratio of the D50% value in the presence of sunscreen to that of the vehicle alone . In either experimental conditions, the D50% in the presence of sunscreens was significantly higher (p < 0.01) than that obtained with the vehicle, demonstrating the sunscreen immunoprotective effect . IPF values obtained with either DC suspensions were very similar and quite comparable to those previously obtained in the skin explant model . Thus, the present in vitro model provides easy tools to determine a new important biological parameter for sunscreens, i.e . immune protection.

Mutat Res, 2004 Mar 14, 558(1-2), 27 - 34
DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field; Chemeris NK et al.; Till the present time, the genotoxic effects of high peak-power pulsed electromagnetic fields (HPPP EMF) on cultured cells have not been studied . We investigated possible genotoxic effects of HPPP EMF (8.8 GHz, 180 ns pulse width, peak power 65 kW, repetition rate 50 Hz) on erythrocytes of the frog Xenopus laevis . We used the alkaline comet assay, which is a highly sensitive method to assess DNA single-strand breaks and alkali-labile lesions . Blood samples were exposed to HPPP EMF for 40 min in rectangular wave guide . The specific absorption rate (SAR) calculated from temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg) . The temperature rise in the blood samples at steady state was 3.5 +/- 0.1 degrees C . The data show that the increase in DNA damage after exposure of erythrocytes to HPPP EMF was induced by the rise in temperature in the exposed cell suspension . This was confirmed in experiments in which cells were incubated for 40 min under the corresponding temperature conditions . The results allow us to conclude that HPPP EMF-exposure at the given modality did not cause any a-thermal genotoxic effect on frog erythrocytes in vitro.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004 Mar, 97(3), 393 - 7
SEM observations of the attachment of human periodontal ligament fibroblasts to non-demineralized dentin surface in vitro; Al-Nazhan S; PURPOSE: The purpose of this investigation was to study in-vitro the attachment behavior of human periodontal ligament (HPDL) fibroblasts to nondemineralized dentin surface using scanning electron microscope . STUDY DESIGN: Thirty root slices of freshly extracted human teeth of 4 mm thickness as well as six 5x5 mm glass slides used as a control were used in this study . The dentin surface of the root slices was not treated with any chemicals to remove the smear layer . The root slices and the glass slides were placed in tissue culture clusters and an amount of 1 ml of HPDL fibroblast cell suspension was placed over the dentin surface of the root slices and the glass slides . They were then placed into an incubator at 37 degrees C and 100% humidity for 4, 24, and 72 hours . At the end of the incubation, the cells were fixed with glutaraldehyde and examined microscopically . RESULTS: Different shapes of fully spread cells were seen . The cells were attached firmly to the dentin surface by the cytoplasmic extension of the lamellipodia and microvilli which were seen extending inside the opening of the dentinal tubules . CONCLUSION: It was concluded that the human dentin surface provided an excellent surface for attachment of periodontal ligament fibroblasts . In addition, the smear layer did not affect the cell attachment.

J Plant Physiol, 2004 Feb, 161(2), 151 - 6
Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ; Stehfest K et al.; A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis . The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1) . First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm . The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.

Haematologica, 2004 Mar, 89(3), 303 - 8
The diagnostic value of CD123 in B-cell disorders with hairy or villous lymphocytes; Del Giudice I et al.; BACKGROUND AND OBJECTIVES: CD123 is an antibody that identifies the a chain of the human interleukin-3 receptor and is expressed in a variety of normal hematopoietic cells, acute leukemia and hairy cell leukemia (HCL) . The aim of the study was to investigate the diagnostic value of CD123 expression in B-cell disorders with circulating hairy and villous lymphocytes . DESIGN AND METHODS: We investigated the diagnostic value of CD123 expression in neoplastic cells from 59 patients with B-cell disorders with circulating hairy or villous lymphocytes: HCL (n=24), the variant form of HCL (n=11) and splenic lymphoma with villous lymphocytes (SLVL) (n=24) . Cells from 12 patients with chronic lymphocytic leukemia were used as controls . Immunophenotypic analysis was performed by flow cytometry on 77 samples from peripheral blood (n=48), bone marrow (n=25) and spleen cell suspensions (n=4) . RESULTS: Our findings show that cells from 95% of typical HCL express CD123 with strong to moderate intensity while this molecule is absent in circulating cells from most cases of HCL-variant (91%) and SLVL (97%) . INTERPRETATION AND CONCLUSIONS: We conclude that CD123 is a useful new marker for distinguishing B-cell disorders with circulating villous lymphocytes as its expression is characteristic of typical HCL with high sensitivity and specificity . However CD123 does not allow the distinction between HCL-variant and SLVL, as both are CD123 negative.

Z Naturforsch {C}, 2004 Jan-Feb, 59(1-2), 48 - 54
Secondary metabolite content in Fabiana imbricata plants and in vitro cultures; Schmeda-Hirschmann G et al.; A rapid in vitro propagation system leading to the formation of shoots, calli, roots, cell suspensions and plantlets was developed for the Andean medicinal plant Fabiana imbricata (Solanaceae) . Massive propagation of shoots and roots was achieved by the temporary immersion system (TIS), morphogenesis and maintenance of cell suspensions by standard in vitro culture techniques . Oleanolic acid (OA), rutin, chlorogenic acid (CA) and scopoletin content in aerial parts of wild growing Fabiana imbricata plants as well as in plantlets regenerated in vitro, callus cultures, cell suspensions and biomass, obtained by the TIS system was assessed by HPLC . On a dry weight basis, the OA content in the aerial parts of the plant ranged between 2.26 and 3.47% while in vitro plantlets, callus and root cultures presented values ranging from not detected up to 0.14% . The rutin content of the samples presented a similar trend with maxima between 0.99 and 3.35% for the aerial parts of the plants to 0.02 to 0.20% for plantlets, 0.12% for cell suspensions and 0.28% for callus . Rutin was not detected in the roots grown by the TIS principle . The CA and scopoletin content in the aerial parts of F . imbricata ranged between 0.22-1.15 and < 0.01-0.55%, respectively . In the plantlets, the concentration of CA was 0.29 to 1.48% with scopoletin in the range 0.09 to 0.64% while in the callus sample, the CA and scopoletin content were 0.46 and 0.66%, respectively . A very different result was found in roots grown by TIS, where both OA and rutin were not detected and its main secondary metabolite, scopoletin was found between a range of 0.99 and 1.41% with CA between of 0.11 and 0.42%.

Methods Mol Biol, 2004, 252, 533 - 43
RNAi expression vectors in plant cells; Akashi H et al.; Suppression by double-stranded RNA (dsRNA) of expression of a target gene is known as RNA interference (RNAi) . Tobacco BY-2 cell suspension has been used as a model cultured plant cell, because it is possible to produce populations of tobacco BY-2 cell suspensions that are uniform and divide synchronously for functional gene analysis . Here, we describe a method to induce RNAi by introducing a hairpin-type dsRNA expression vector into BY-2 cells via electroporation . This methodology should facilitate the analysis of individual gene function in plant cells.

Fundam Clin Pharmacol, 2003 Dec, 17(6), 709 - 15
Direct demonstration of nitric oxide formation in organs of rabbits treated by transdermal glyceryl trinitrate using an in vivo spin trapping technique; Clermont G et al.; Glyceryl trinitrate (GTN) is commonly delivered by a patch for the treatment of angina pectoris . The idea is now generally accepted that GTN requires a biotransformation process that activates the drug, in particular through nitric oxide (NO) generation . However, the pharmacokinetics of NO delivery from GTN still remains obscure . The objective of this study was to assess GTN-derived NO formation in vascular tissues and organs in rabbit given GTN patches . NO levels were evaluated in rabbits after 3 h of treatment with a 10 mg GTN patch (GTN group; n = 7) or a placebo patch (CTL; n = 7) . Nitrosylhaemoglobin (HbNO) was evaluated by electron spin resonance (ESR) spectroscopy in red cell suspension . In vivo spin trapping technique using FeMGD as a spin trap, associated with ESR was used to quantify NO in tissues . The NO-spin trap complex, which is a relatively stable product, has been measured in several tissues . The ESR spectrum corresponding to HbNO was not found in red cell of GTN or CTL rabbits . The spectrum corresponding to the NO-spin trap complex was observed in all analysed tissues of CTL rabbits . The signal was significantly increased in liver, renal medulla, heart left ventricle and spleen of GTN-treated rabbits, and to a lesser extent in right ventricle and lung . No difference was shown between NO-spin trap levels measured in aorta or inferior vena cava from GTN or CTL rabbits . These data suggest that GTN patch treatment induced NO release, and that tissue-specific differences in transdermal GTN-derived NO exist . The GTN-NO pathway appears to be largely involved in organs such as the liver, kidney and heart.

Anticancer Drugs, 2004 Mar, 15(3), 265 - 8
Dysregulation of protein kinase C activity in chemoresistant metastatic breast cancer cells; Schondorf T et al.; This study was performed to evaluate the role of protein kinase C (PKC) activity in the development of chemoresistance in clinical breast cancer cells . To simulate the clinical situation, native tumor cells derived from 10 patients with advanced breast cancer were brought into short-term cultures, and treated with anthracyclines (doxorubicin, mitoxantrone), paclitaxel and combinations, respectively . After 3 days of incubation, we determined total PKC activity relative to each control incubated with blank medium . Furthermore, we determined the chemoresistance against these drugs from each cell population separately . Relative PKC activity ranged from 14 to 249%; 64% (37 of 58) of the breast cancer cell suspensions were considered chemoresistant . There was a non-significant trend to a higher relative PKC activity in resistant cells compared to non-resistant cells (p=0.058), regardless of the antineoplastic agent investigated . The individual variability in both PKC activity and chemoresistance pattern revealed that dysregulated PKC activity mediates resistance to antineoplastics . In order to achieve clinical value, evaluation of more data concerning the PKC signal-transduction pathway is necessary . New protocols of cancer treatment will require this information in order to be successful.

J Orthop Res, 2004 Mar, 22(2), 353 - 61
Osteoblast-like cell adhesion to bone sialoprotein peptides; Rapuano BE et al.; A number of studies have suggested that biomimetic peptides can be used in the design of a new generation of prosthetic implants to promote the successful biointegration of the implant materials . In the current study, the in vitro bioactivities of several peptides representing RGD (Arg-Gly-Asp)-containing sequences of bone sialoprotein (BSP) toward an osteoblast-like cell line (MC3T3-E1) were examined to provide insight into the molecular basis of BSP's interaction with bone cells . BSP residues 283-288, 281-290, 278-293 and 278-302 were coated on polystyrene surfaces in 96-well non-tissue (untreated) culture plates, and their osteoblast adhesive properties compared to intact BSP and fibronectin as positive controls . BSP peptides 278-302 and 278-293 were found to be the most potent in their adhesive activity, increasing the number of adherent cells to 350% of control levels at an added concentration of 1 microM . Since these two peptides were equivalent in potency, it is suggested that the region 294-302 beyond the RGD domain is not necessary for cell binding . In comparison, peptides 283-288 and 281-290 were only active at concentrations greater than 200 microM . 50-70% of the peptide-stimulated adhesion was inhibited by the pretreatment of cell suspensions with solution phase RGD, suggesting that a portion of the peptides' adhesive effects was specific and integrin-mediated, although other non-RGD flanking regions were probably also involved in the mechanism of adhesion . Importantly, a modified BSP peptide, in which an aspartic acid residue at position 288 of the RGD sequence was replaced by a glutamic acid residue to form RGE, was completely inactive as a cell adhesion stimulus at concentrations up to 200 microM . Thus, despite the potential role of non-RGD flanking regions, an intact RGD tripeptide was essential for all of the adhesive activity of the BSP peptides.

Nephrology (Carlton), 2003 Jun, 8(3), 150 - 155
Effects of bicarbonate buffered dialysate on human peritoneal mesothelial cell intracellular calcium homeostasis; Bird SD et al.; This study compares the biocompatibility of two bicarbonate-based peritoneal dialysis (PD) solutions using the measurement of intracellular free calcium (Ca(i)(2+)) as a sensitive parameter of cell function in human peritoneal mesothelial cells (hPMC) . Fura-2-loaded hPMC suspensions were exposed to bicarbonate (38 mmol/L) and bicarbonate (25 mmol/L), lactate-buffered PD (15 mmol/L) solutions at pH 7.4 and compared with Krebs-Ringer physiological saline (KRS; pH = 7.4) . Resting Ca(i)(2+) values and 4br-A23187 (1.0 micro mol/L) induced transients were compared in treatment and control groups . In separate studies, the effect that low saline pH had on Ca(i)(2+) homeostasis was examined . Suspended cells or cells attached to coverslips were bathed in citric acid-phosphate (McIllvaine's) buffered saline (MBS, pH = 7.4) . Cells were acidified (pH = 5.3) with citric acid and then challenged with ionophore . Ionophore challenge produced a significantly reduced Ca(i)(2+) transient response in cells exposed to the bicarbonate/lactate fluid compared with bicarbonate or KRS . Acidified cell suspensions produced a small monophasic Ca(i)(2+) transient rise that was short lived . Gradual recovery of MBS to pH 7.4 produced no changes to Ca(i)(2+) homeostasis of cell monolayers . Ionophore treatment produced a biphasic response identical to cells bathed in KRS . This study has demonstrated that short-term exposure to bicarbonate did not alter Ca(i)(2+) homeostasis directly, or subsequent modulation of intracellular pH . The MBS system provided a reliable method of modifying the external pH during continuous Ca(i)(2+) measurement.

Phys Med Biol, 2004 Feb 7, 49(3), 359 - 69
The use of chloroaluminium phthalocyanine tetrasulfonate (AlPcTS) for time-delayed fluorescence imaging; Gundy S et al.; Phthalocyanine derivatives are currently under investigation for use in photodynamic therapy, which is a promising cancer treatment . These materials, which display preferential uptake in cancerous cells, also exhibit high fluorescence yields and can be used for tumour detection . Problems with steady-state fluorescence techniques such as excitation scatter and background autofluorescence can be eliminated by using time-resolved imaging techniques without the need for filters . A tissue phantom was assembled to test a constructed time-gated imaging system by drilling 36 wells of varying diameter and depth (10 mm to 1 mm) into a block of polymethyl methacrylate (PMMA) . The system was used to record images of chloroaluminium phthalocyanine tetrasulfonate (AlPcTS) at differing concentrations in neat aqueous solvent and cell suspensions within the wells . A mixture of Intralipid (to mimic tissue scatter) and Evan's blue (to mimic tissue absorption) of depths ranging from 1 mm to 10 mm was placed on top of the PMMA block . The ensuing images were analysed using signal-to-noise ratios and contrast-detail curves . The results indicate that the time-gated imaging system can prevent background excitation scatter from distorting the fluorescence signal from a longer-lived photosensitizer without the need for filters.

Plant Mol Biol, 2003 Nov, 53(4), 423 - 42
Genome-wide gene expression in an Arabidopsis cell suspension; Menges M et al.; Plant cell suspension cultures are invaluable models for the study of cellular processes . Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays . Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system . Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes . Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony . We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method.

J Asian Nat Prod Res, 2004 Jun, 6(2), 93 - 7
Biotransformation of triptolide and triptonide by cell suspension cultures of Catharanthus roseus; Ning LL et al.; Catharanthus roseus cell suspension cultures were used to bioconvert both triptolide (1) and triptonide (2) . The same reaction path was followed in both biotransformations . Two biotransformed products were obtained and their structures identified as triptriolide (3) and 12beta,13alpha-dihydroxytriptonide (4), respectively, from 1 and 2 . Product 4 is a new compound.

Lab Chip, 2004 Feb, 4(1), 47 - 52 Epub 2003 Oct 29.
Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip; Gao J et al.; A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip . Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design . The effective separation channel length was 35 mm . During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing . Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow . A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel . Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates . The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations . Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells . NDA derivatized GSH was detected using LIF . A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells . The average cellular concentration of GSH in human erythrocytes was found to be 7.2 {times} 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell) . The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.

Oral Oncol, 2004 May, 40(5), 474 - 82
Delivery of (10)boron to oral squamous cell carcinoma using boronophenylalanine and borocaptate sodium for boron neutron capture therapy; Obayashi S et al.; Boron neutron capture therapy (BNCT) is a unique radiation therapy in which boron compounds are trapped into tumor cells . To determine the biodistribution of boronophenylalanine (BPA) in nude mice carrying oral squamous cell carcinoma (SCC), BPA was administered at a dose of 250 mg/kg body weight intraperitoneally . Two hours later, (10)B concentration in the tumor was 15.96 ppm and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 6.44, 4.19, 4.68 and 4.56, respectively . Two hours after the administration of borocaptate sodium (BSH) at a dose of 75 mg/kg body weight, (10)B concentration in the tumor was 3.61 ppm, and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 0.77, 1.05, 0.60 and 0.59, respectively . When cultured oral SCC cells were incubated with BPA or BSH for 2 h and then exposed to thermal neutrons, the proportion of survival cells that were capable of forming cell colonies decreased exponentially, depending on (10)B concentration . BPA-mediated BNCT was more efficient than BSH-mediated BNCT . Addition of boron compounds in the cell suspension during neutron irradiation enhanced the cell-killing effect of the neutrons . These results indicate that BPA is more selectively incorporated into human oral SCC as compared with normal oral tissues, and that both extra- and intra-cellular BPA contribute to the cell-killing effect of BNCT . BPA may be a useful boron carrier for BNCT in the treatment of advanced oral SCC.

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