Cell suspensions

Phytochemistry, 2000 Sep, 55(1), 51 - 7
Antifungal monoterpene production in elicited cell suspension cultures of Piqueria trinervia; Saad I et al.; Cell suspension cultures of the traditional medicinal plant Piqueria trinervia Cav., which synthesizes monoterpene piquerol A, were established . A defense response was induced in the cultures when eight homogenized fungi isolated from wild populations of P . trinervia were added . Piquerol A was not produced in the elicited system, while four other substances were synthesized de novo . They were excreted into the medium and inhibited in vitro fungal growth . The most abundant substance produced in this system was a new monoterpene: 2-methylene-7,7-dimethylbicyclo (3,3,1) heptane-4,6-diol . Monoterpenes in the cell suspension culture reported here were produced via two metabolic channels: the first acted constitutively and expressed in liquid and solid cultures, the second is inducible in response to several pathogens and elicitor substances.

FEBS Lett, 2000 Sep 29, 482(1-2), 125 - 30
Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells; Seregelyes C et al.; We have isolated a 483-bp-long full-length cDNA clone encoding a non-symbiotic hemoglobin called Mhb1, the first one found in alfalfa . This non-symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome . The Mhb1 mRNA was found only in the roots of alfalfa plants . The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment . The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture . The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm . A potential link to the nitric oxide signalling pathway is also discussed.

Biochim Biophys Acta, 2000 Sep 27, 1487(2-3), 113 - 27
Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo; Kamat JP et al.; Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties . Its antioxidant ability, however, has not been examined in detail . Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions . Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls . Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances . The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase . ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride . Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM . The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol . When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis . To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen . The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1) . In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.

Hum Pathol, 2000 Sep, 31(9), 1051 - 4
Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping; Bavikatty NR et al.; CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML) . However, until recently, analysis for CD10 has generally required fresh or frozen tissue . 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue . Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL . CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias . The results matched in 100% (20 of 20) of AML . Five percent (1 of 20) of AMLs expressed CD10 . Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry . CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL . Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry . In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry . We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia . This technique is useful in distinguishing AML from ALL.

Arthritis Rheum, 2000 Sep, 43(9), 2046 - 55
Mesenchymal cells expressing bone morphogenetic protein receptors are present in the rheumatoid arthritis joint; Marinova-Mutafchieva L et al.; OBJECTIVE: To evaluate the presence of cells of an early mesenchymal lineage, as judged by the expression of bone morphogenetic protein receptors (BMPRs), in the joints of normal individuals and patients with rheumatoid arthritis (RA) . METHODS: Synovial fluids, single cell suspensions of cultured fibroblast-like synoviocytes (FLS), and synovial tissues were examined by immunohistology with antibodies to BMPR type IA (BMPRIA), BMPRIB, and BMPRII and then quantified using computerized image analysis . Other antibodies were evaluated by cytofluorography . RESULTS: In primary cultures of joint effusions from patients with RA and other forms of inflammatory arthritis, there were large adherent cells with the appearance of either fibroblasts or stromal cells that stained with antibodies to mesenchymal elements-CD44, type I collagen, alpha-actin, and vimentin-but not with antibodies to hematopoietic markers . These cells proliferated rapidly, expressed BMPRIA and BMPRII, and soon became the predominant cells in culture . They were retained through multiple passages and persistently displayed surface vascular cell adhesion molecule 1 . Immunohistochemical analysis of cultured RA FLS (passages 3, 4, and 6; n = 6) revealed that 11.6% were BMPR-positive, while only 2.0% of osteoarthritis FLS (passage 4; n = 3) were BMPR-positive, and 1 normal synovial culture had no BMPR-positive cells . In all RA synovial membranes examined (n = 9), BMPRI- and BMPRII-expressing cells were identified in the intimal lining and were also scattered in the subintima . These cells constituted approximately 25% and approximately 7% of the cells in each area, respectively . Double immunostaining showed no coexpression of BMPR-positive cells with CD68, CD34, or CD3 . Cells expressing BMPR were not seen in any normal synovial samples (n = 4) . Strong staining for BMPR was identified on cells at the invasive front of the pannus and at sites of cartilage erosion . CONCLUSION: The inflamed RA joint contains BMPR-positive mesenchymal cells . Their origin is still speculative, but since their counterparts in the bone marrow are essential for osteoclastogenesis, support lymphocyte development and maturation, and protect T cells and B cells from programmed cell death, the BMPR-positive cells may be essential elements in the pathogenesis of RA and other inflammatory forms of chronic synovitis.

Exp Cell Res, 2000 Oct 10, 260(1), 146 - 59
Stem cell characteristics of transplanted rat mammary clonogens; Kim ND et al.; Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials . When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient . Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens . Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition . The current experiments were designed to test these expectations . The data are discussed in the context of results of previous studies with this and other experimental models . The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells .

Dig Dis Sci, 2000 Aug, 45(8), 1631 - 8
Reaggregation of rat dissociated myenteric plexus in extracellular matrix gels; Schafer KH et al.; The aim of this study was to investigate the growth behavior of freshly dissociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurotrophic factor (GDNF) . Therefore, cell suspensions of the dissected myenteric plexus of newborn rats were cultured in freshly prepared gels of commercially available mixtures of collagen, laminin, and hepatoglycans as a first step towards mimicking the natural environment of the myenteric plexus . The cultures were kept either in chemically defined serum-free medium alone or supplemented with GDNF . Cultures on polylysinc-coated glass cover slips served as controls . Dissociated myenteric plexus grown on polylysine formed dense clusters of neurons with radially outgrowing nerve fibers, while the neurons cultured in the gel reaggregated to much smaller clusters . These contained, depending on the culture conditions, 2-10 neurons . The morphology of the network that was seen in the gels after a few days in vitro resembled very closely the in situ situation of the submucous plexus and the myenteric plexus in hypoganglionic children . Electron microscope investigations showed a high degree of organization with fiber bundles and vesicle-containing varicosities and growth cones . Independent of the method of culturing, GDNF obviously influenced the growth behavior of the dissociated plexus . The size of the ganglia was larger, and the secondary network denser when GDNF was supplemented . Moreover, the enteric neurons in the gel cultures tended to be larger in size when treated with GDNF . Three-dimensional cultures of dissociated myenteric plexus in an ECM gel might be a valuable tool towards the understanding of the formation of the enteric nervous system during development, especially considering pathological conditions such as Hirschsprung's disease or other dysganglionic diseases.

Pflugers Arch, 2000, 440(5 Suppl), R193 - 4
Effect of pH on red blood cell deformability; Kuzman D et al.; The effect of pH on the red blood cell (RBC) deformability, which is a consequence of a change of cell membrane elastic properties is studied experimentally . With the intention to reduce the effects on deformability of cell geometry and cytoplasmic viscosity, we measured the deformability of the cells with the same volume at various pH of cell suspension from 6.2 to 8.0 . Constant cell volume was achieved by varying osmolarity . Deformability was quantified by measuring the elongation of RBCs subjected to velocity gradient in a transparent cone-plate rheoscope . Observed significant decrease of deformability at lower pH leads to the conclusion that membrane elastic properties could be affected by pH changes in the range from 6.2 to 8.0.

Pflugers Arch, 2000, 440(5 Suppl), R49 - 50
In vitro functional tests for evaluation of stimulating capacity of cultured human dendritic cells; Hajdinjak T et al.; Basic functional test for evaluation of in vitro cultured human dendritic cells (DC) is primary allogeneic one-way mixed lymphocyte reaction (MLR) . In this way, one can evaluate stimulating capacity, which is a basic characteristic of DC . The proliferation of cells is measured through incorporation of 3H-thymidine . Normally proliferation is measured at days 5-7 . We studied kinetics of proliferative responses initiated with different stimulating cell suspensions to evaluate differences and possibly reduce time needed to perform this test . Gradual increase in response from days 1 to 7 and a significant difference from controls (peripheral blood mononuclear cells) seen from day 4 was noted if macrophages were used as stimulators . A consistently higher proliferation, compared to controls, was always found already on day 2 when mature DC were used as stimulators . The reaction peaked 2 to 3 days earlier and was also more than two times more intense . This maximal and significantly higher response, consistently seen already after 48 hours, allows us to confirm the presence of mature DC in stimulating suspensions much earlier than previously.

Plant J, 2000 Sep, 23(6), 785 - 94
Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity; Orvar BL et al.; Many plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non-freezing temperatures at the onset of winter . CA is associated with gene expression that requires transient calcium influx into the cytosol . Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold-acclimatize . Conversely, chemical agents causing increased calcium influx induce cold acclimatization-specific (cas) gene expression in alfalfa at 25 degrees C . How low temperature triggers calcium influx is, however, unknown . We report here that induction of a CA-specific gene (cas30), calcium influx and freezing tolerance at 4 degrees C are all prevented by cell membrane fluidization, but, conversely, are induced at 25 degrees C by membrane rigidification . cas30 expression and calcium influx at 4 degrees C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25 degrees C by the actin microfilament destabilizer cytochalasin D (CD) . JK blocked the membrane rigidifier-induced, but not the calcium channel agonist-induced cas30 expression at 25 degrees C . These findings indicate that cytoskeleton re-organization is an integral component in low-temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidification and calcium influx in CA.

J Invest Dermatol, 2000 Oct, 115(4), 680 - 6
Magnesium ions inhibit the antigen-presenting function of human epidermal Langerhans cells in vivo and in vitro . Involvement of ATPase, HLA-DR, B7 molecules, and cytokines; Schempp CM et al.; The combination of seawater baths and solar radiation at the Dead Sea is known as an effective treatment for patients with psoriasis and atopic dermatitis . Dead Sea water is particularly rich in magnesium ions . In this study we wished to determine the effects of magnesium ions on the capacity of human epidermal Langerhans cells to stimulate the proliferation of alloreactive T cells . Twelve subjects were exposed on four subsequent days on the volar aspects of their forearms to 5% MgCl2, 5% NaCl, ultraviolet B (1 minimal erythemal dose), MgCl2 + ultraviolet B, and NaCl + ultraviolet B . Epidermal sheets were prepared from punch biopsies and were stained for ATPase and HLA-DR . Compared with untreated skin, the number of ATPase+/HLA-DR+ Langerhans cells was significantly reduced after treatment with MgCl2 (p = 0.0063) or ultraviolet B (p = 0.0005), but not after NaCl (p = 0.7744) . We next questioned whether this reduced expression of ATPase and HLA-DR on Langerhans cells bears a functional relevance . Six subjects were treated on four subsequent days with 5% MgCl2, ultraviolet B (1 minimal erythemal dose), and MgCl2 + ultraviolet B . Epidermal cell suspensions from treated and untreated skin were assessed for their antigen-presenting capacity in a mixed epidermal lymphocyte reaction with allogeneic naive resting T cells as responder cells . Treatment with MgCl2, similarly to ultraviolet B, significantly reduced the capacity of epidermal cells to activate allogeneic T cells (p = 0.0356) . Magnesium ions also suppressed Langerhans cells function when added to epidermal cell suspensions in vitro . The reduced antigen-presenting capacity of Langerhans cells after treatment with MgCl2 was associated with a reduced expression by Langerhans cells of HLA-DR and costimulatory B7 molecules, and with a suppression of the constitutive tumor necrosis factor-alpha production by epidermal cells in vitro . These findings demonstrate that magnesium ions specifically inhibit the antigen-presenting capacity of Langerhans cells and may thus contribute to the efficacy of Dead Sea water in the treatment of inflammatory skin diseases.

Eur J Ultrasound, 2000 Sep, 12(1), 81 - 8
Mechanical properties of bovine aortic endothelial cells in suspension studied by ultrasonic interferometry; Boynard M et al.; OBJECTIVE: Cell adhesion phenomenon has been extensively studied in the last decade and was shown to be mediated by specialized molecules and driven by physical forces . Cohesion of the vessel wall cells is also dependent on adhesion molecules but less is known about the physical forces involved . To investigate endothelial cell/endothelial cell interaction from a mechanical point of view, we have used an ultrasonic interferometry device, named EchoCell, which has been previously designed to study red blood cell-red bood cell (RBC-RBC) interaction . METHODS: Bovine aortic endothelial (BAE) cells were cultured, detached, then suspended in buffer and their mechanical and geometrical properties studied with the EchoCell system . The ultrasonic apparatus measures both the accumulation rate of cells in suspension on a solid plate and the acoustical impedances of the suspension and the sediment . RESULTS: In suspension, BAE exhibited, in our experimental conditions (3x10(6) cells per ml), a spherical size evaluated by calculation at a mean radius of 7+/-2 microm . Moreover, no BAE aggregation occurred at the concentrations used . The acoustical impedance of the BAE suspensions calculated from all the samples studied, in the cell concentration range from 1.5x10(6) to 6x10(6) cells per ml, was 1.52x10(6) Rayl (kg m(-2) s(-1)) . Furthermore, the acoustical impedance of the cell sediment was found to be independent on the initial cell suspension concentration and equal to 1.63x10(6) Rayl (kg m(-2) s(-1)) . Estimation of the volume fraction of BAE inside the sediment allows to evaluate the ultrasonic velocity and the elastic bulk modulus of cells . CONCLUSION: The ultrasonic interferometry method appears particularly interesting to study geometrical and mechanical (acoustical impedance, sound velocity, elastic bulk modulus) properties of BAE cells.

Ultrasound Med Biol, 2000 Jul, 26(6), 1043 - 9
High-frequency backscatter and attenuation measurements of selected bovine tissues between 10 and 30 MHz; Maruvada S et al.; There are now diagnostic ultrasonic imaging devices that operate at very high frequencies (VHF) of 20 MHz and beyond for clinical applications in ophthalmology, dermatology, vascular surgery, endoluminal imaging and small animal imaging . To be able to better interpret these images and to further the development of these devices, knowledge of ultrasonic attenuation and scattering of biological tissues in this frequency range is crucial . Attenuation and backscatter coefficients (BSCs) of bovine tissues in the frequency range of 10 to 30 MHz were measured, respectively, using a standard substitution method for attenuation measurements and a modified narrow-band substitution method for scattering measurements . A modified substitution method for scattering measurements has to be used at high frequencies because unfocused transducers due to their decreased sensitivity cannot be used in the simple substitution method . In the modified method, the flat reflector is substituted by a particulate reference medium whose BSC is well-known and documented; in this case, a red cell suspension . In this paper, experimental results on BSC and attenuation coefficient measured between 10 and 30 MHz are reported . The frequency dependence of backscatter of the selected bovine tissues ranges from 2.4 to 3.5, whereas attenuation is observed to be still approximately linearly proportional to frequency . The BSC measured with the modified method is in good agreement with those obtained with the standard method between 10 and 20 MHz.

Plant Sci, 2000 Sep 8, 158(1-2), 129 - 137
Decrease in the regeneration potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration by the auxin transport inhibitor, 2,3,5-triiodobenzoic acid; Nakano M et al.; Cell suspension cultures of Lilium formosanum Wallace were initiated from bulb scale-derived calli and subcultured every 2 weeks using a medium containing 5 microM 4-amino-3,5,6-trichloropicolinic acid (picloram) . Almost all cell clumps from the suspension cultures developed numerous somatic embryos following their transfer onto a plant growth regulator-free medium, while they vigorously produced shoot buds on media containing 0.5 or 5 microM 6-benzyladenine (BA) . The high regeneration potential on a plant growth regulator-free medium was maintained for up to 54 months, but it gradually decreased thereafter, and only a few adventitious shoots and embryos were obtained from 75-month-old cultures . For restoring the regeneration potential of these cultures, various treatments with plant growth regulators were applied, among which about 10-fold increases in the number of regenerated shoot buds were obtained with 0.5 or 5 microM 2,3,5-triiodobenzoic acid (TIBA) in combination with 0.5 or 5 microM BA or N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea (thidiazuron) . Only shoot buds were produced from the cell clumps cultured on TIBA-containing media, and these shoot buds developed into complete plantlets after they were excised from the calli and transferred to a plant growth regulator-free medium.

Plant Sci, 2000 Sep 8, 158(1-2), 41 - 51
Oligosaccharides potentiate methyl jasmonate-induced production of paclitaxel in Taxus canadensis; Linden JC et al.; The interdependence of methyl jasmonate (MJ) with chitin and chitosan derived elicitors in formation of paclitaxel was studied using plant cell suspension cultures of Taxus canadensis . Induction of paclitaxel biosynthesis was enhanced when MJ and elicitors were added 8 days after culture transfer compared to treatments in which only MJ or only elicitors were added . The enhancement of the paclitaxel biosynthesis response to MJ concentration was roughly linear between 0 and 200 microM using colloidal chitin or oligosaccharides of chitin and chitosan as elicitors . MJ concentrations greater than 200 microM were inhibitory . In kinetic studies, culture growth and substrate utilization were inhibited when the cultures were elicited with 100 microM MJ and with 0.63 mg l(-1) N-acetylchitohexaose and with 100 microM MJ alone; paclitaxel yields were 10-fold greater under the latter condition than the former . Ethylene biosynthesis by the cell cultures in response to elicitation is implicated in regulation of the response.

Prikl Biokhim Mikrobiol, 2000 Jul-Aug, 36(4), 422 - 7
{Proteolytic activity of the enzyme complex of the mammalian pancreas in comparison with pancreatin}; Berdutina AV et al.; The proteolytic activity and thermal stability of the enzyme complex of cell suspension from pig and bovine pancreas glands was compared with those of pancreatin . The enzyme complex displayed the highest thermal stability and activity at 50 degrees C . The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 +/- 0.1) at which this complex had the maximum proteolytic activity were determined . Pancreatin had a pH optimum of 8.0 +/- 0.1.

Exp Neurol, 2000 Oct, 165(2), 268 - 77
Time course of apoptotic cell death within mesencephalic cell suspension grafts: implications for improving grafted dopamine neuron survival; Sortwell CE et al.; The vast majority ( congruent with 90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum . Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation . This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts . Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively . Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum . Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation . However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days . No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found . Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis .

Photochem Photobiol, 2000 Sep, 72(3), 320 - 6
A novel express bioassay for detecting toxic substances in water by recording rhodopsin-mediated photoelectric responses in Chlamydomonas cell suspensions; Govorunova EG et al.; The influence of Cu2+, Zn2+, Cd2+, Pb2+ and formaldehyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was investigated using three modifications of a recently developed population method for electrical recording (in nonoriented, phototactically preoriented (PO) and gravitactically preoriented cell suspensions) . The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the responses . Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simultaneously with an inhibition of their photoelectric cascade, photo-orientation and motility . The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive phototaxis in the presence of the toxic substances . Of the two major components of the photoelectric cascade, the regenerative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the photoreceptor current . The results obtained show that measurement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bioassay for testing environmental pollutants in water samples.

Planta, 2000 Aug, 211(3), 430 - 9
Molecular characterization of the expression of distinct classes of cyclins during the early development of tomato fruit; Joubes J et al.; Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds from two distinct phases of growth, essentially cell division and cell expansion . In this study, we investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins, during tomato fruit development . We isolated six genes designated Lyces;CycA1;1, Lyces;CycA2;1, Lyces; CycA3;1, Lyces;CycB1:1 and Lyces;CycB2;1 encoding tomato mitotic cyclins, and Lyces;CycD3;1 encoding a G1 cyclin . The accumulation of transcripts was predominantly associated with mitotically active organs: developing fruits, young leaves and roots, and with cell-suspension cultures under appropriate sugar feeding conditions . Transcripts for all the isolated cyclin genes could be detected in the epidermis and pericarp of fruit tissues where some slight mitotic activity still remained at the onset of ripening . However, Lyces;CycA3;1 and Lyces;CycD3;1 were expressed in the gel tissue at the late stage of fruit development, suggesting that they are involved in endoreduplication of the differentiated and giant cells of the gel tissue.

J Immunol Methods, 2000 Aug 28, 242(1-2), 21 - 31
Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization; Curry MP et al.; Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs . To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays . The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue . The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects . Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques . A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I . These cells were suitable for phenotypic characterisation by flow cytometry . They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines.

Med Biol Eng Comput, 2000 Jul, 38(4), 384 - 9
Derivation of extracellular fluid volume fraction and equivalent dielectric constant of the cell membrane from dielectric properties of the human body . Part 2: A preliminary study for tracking the progression of surgical tissue injury; Tatara T et al.; A study is conducted to determine whether the extracellular fluid (ECF) volume fraction and equivalent dielectric constant of the cell membrane epsilon m, derived from the dielectric properties of the human body can track the progression of surgical tissue injury . Frequency-dependent dielectric constants and electrical conductivities of body segments are obtained at surgical (trunk) and non-surgical sites (arm and leg) from five patients who have undergone oesophageal resections, before and at the end of surgery and on the day after the operation . The ECF volume fraction and the equivalent epsilon m of body segments are estimated by fitting the dielectric data for body segments to the cell suspension model incorporating fat tissue, and their time-course changes are compared between body segments . By the day after the operation, the estimated ECF volume fraction has increased in all body segments compared with that before surgery, by 0.13 in the arm, 0.16 in the trunk and 0.14 in the leg (p < 0.05), indicating postoperative fluid accumulation in the extracellular space . In contrast, the estimated equivalent epsilon m shows a different time course between body segments on the day after the operation, characterised by a higher change ratio of epsilon m of the trunk (1.34 +/- 0.66, p < 0.05), from that of the arm (0.66 +/- 0.34) and leg (0.61 +/- 0.11) . The results suggest that the equivalent epsilon m of a body segment at a surgical site can track pathophysiological cell changes following surgical tissue injury.

Med Biol Eng Comput, 2000 Jul, 38(4), 377 - 83
Derivation of extracellular fluid volume fraction and equivalent dielectric constant of the cell membrane from dielectric properties of the human body . Part 1: Incorporation of fat tissue into cell suspension model in the arm; Tatara T et al.; The non-invasive characterisation of cell pathophysiology is clinically important . A cell suspension model is applied to derive the extracellular fluid (ECF) volume fraction and the equivalent dielectric constant of the cell membrane epsilon m from the dielectric properties of human arms . Frequency-dependent dielectric constants and electrical conductivities of arms are obtained from 35 surgical patients over a frequency range of 5-1000 kHz . The cell suspension model is applied to fit the data using a complex non-linear least-squares method . The arms show typical dielectric dispersions, although the cell suspension model yields a poor fitting in dielectric constants at lower frequencies and electrical conductivities at higher frequencies . In contrast, a new cell suspension model, taking into account the fat tissue component, remarkably improves the overall fitting performance, allowing estimation of the volume fractions of ECF (0.34 +/- 0.05) and fat tissue (0.16 +/- 0.04) and the equivalent epsilon m (23 +/- 9) . The resulting estimates of the volume fraction of fat tissue are in good correlation with arm skinfold thickness (fat volume fraction of arm = 2.42 x 10(-3) x arm skinfold thickness (mm) + 0.099, R = 0.756, p < 0.0001) . Therefore it is concluded that the newly derived cell suspension model is well suited for the description of the dielectric properties of human tissues and thus the derivation of the ECF volume fraction and equivalent epsilon m.

Biocell, 2000 Aug, 24(2), 139 - 44
Cell lines of Taxus species as source of the anticancer drug taxol; Goleniowski ME; Callus culture of Taxus baccata and Taxus x Media were induced using explants of young stems and female gametophyte . Culture conditions have been established for the cell suspension of the different callus cell lines . Callus were induced from Taxus baccata and Taxus x Media using Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4 D (2,4-dichlorophenoxyacetic) and benzylaminopurine (BA) . All the cultures grew slowly following the first subculture and the majority turned brown and ceased growth . The fast growing callus lines constituted a habituated callus lines (CFGTB; CFGTM; CSTB and CSTB) . These callus lines were used to induce cell suspension in the best nutritional medium (1 mg/l 2,4-D and 0.1 mg/l BA) . The callus exhibited levels of taxol ranging from 0.1 to 15 mg/Kg on a dry weight basis . Suspension cultures of Taxus baccata (CSTB and CFGTB) and Taxus x Media (CSTM and CFGTM) were maintained at 25 degrees C on a MS medium with two weeks transfers . The maximum taxol production for suspension cell was within the range 5 to 6 mg/l.

Biocell, 2000 Aug, 24(2), 133 - 8
Peroxidases from cell suspension cultures of Brassica napus; Agostini E et al.; Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators . They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots . Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures . Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme . Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points . These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 179 - 82
{Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion}; Guo WW et al.; Protoplasts isolated from cell suspension cultures of 'Bonnaza' navel orange (Citrus sinensis L . Osbeck) were electrically fused with mesophyll protoplasts of rough lemon (Citrus jambhiri Lush) and Goutou orange (Citrus aurantium L.) respectively . Plants regenerated from both fusion combinations . Chromosome counting of randomly selected fifty two globular embryoids as well as all the regenerated seventy four plants from Bonnaza navel + rough lemon revealed that twenty six embryoids were tetraploids, and the rest were diploids while 100% regenerated plants were tetraploids . The results inferred that somatic hybrids were more competitive than parental genotypes in the process of plant regeneration . All the regenerated 14 plants from Bonnaza navel + Goutou orange were tetraploids as revealed by chromosome counting . POX isozyme and RAPD analysis verified that the plants from Bonnaza navel + rough lemon were hybrids, and RAPD analysis confirmed the hybridity of those from Bonnaza navel + Goutou orange.

Phytochemistry, 2000 Aug, 54(7), 657 - 66
Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis; Warzecha H et al.; Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds . In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides . Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l . Cell cultures do contain a specific glucosidase . known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis . Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli . RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized . The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed . Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.

Phytochemistry, 2000 Aug, 54(7), 649 - 55
Flavanone 8-dimethylallyltransferase in Sophora flavescens cell suspension cultures; Yamamoto H et al.; Dimethylallyl diphosphate: naringenin 8-dimethylallyltransferase (EC 2.5.1) was characterized . The enzyme was enantiospecific for (-)-(2S)-naringenin and utilized 3,3-dimethylallyl diphosphate as sole prenyl donor . It required Mg2+ (optimum concentration, 10 mM), and has an optimum pH of 9-10 . The apparent Km values for 3,3-dimethylallyl diphosphate and naringenin were 120 and 36 microM, respectively . The microsomal fraction prenylated several other flavanones at the C-8 position less effectively as compared with naringenin . Interestingly, when 2'-hydroxynaringenin was used as a prenyl acceptor, the 8-lavandulyl (sophoraflavanone G) and the 6-dimethylallyl derivatives were formed, together with the 8-dimethylallyl derivative, leachianone G . These results suggest that the 2'-hydroxy group of naringenin plays an important role for the formation of a lavandulyl group.

Braz J Med Biol Res, 2000 Sep, 33(9), 1015 - 21
A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system; Yarzabal A et al.; The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied . Plasminogen bound to the surface of H . pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA . Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins . Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension . The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells . epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors . The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established . Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated . These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages.

Cell Transplant, 2000 May-Jun, 9(3), 395 - 407
The morphology, integration, and functional efficacy of striatal grafts differ between cell suspensions and tissue pieces; Watts C et al.; In order to develop a surgical protocol for use in clinical trials of striatal transplantation in Huntington's disease (HD), the issues involved in the preparation and implantation of the embryonic striatal tissue must be addressed . Rodent models of HD offer the best experimental paradigm with which to study various aspects of striatal transplantation . In this article we present the results of an investigation of the role of trypsin and the process of trituration in the preparation of cell suspensions compared to the use of solid pieces of tissue . The embryonic material was derived from the lateral ganglionic eminence (LGE) and implanted into the excitotoxically lesioned striatum of the host rats . Twelve weeks following implantation, retrograde tracing of projections from the graft to the globus pallidus was performed . Grafts derived from cell suspensions triturated in the presence of trypsin contained larger quantities of striatal tissue within the graft and more DARPP-32-positive medium spiny neurons than grafts implanted as fragments of tissue . Afferent and efferent connectivity was also better in the trypsinized suspension graft group . Modest recovery in paw reaching was observed contralateral to the grafted side in animals implanted with solid fragments of embryonic striatal tissue . No relationship was observed between functional effect and the graft anatomy . These results suggest that local graft host interaction may also be involved in graft-mediated functional recovery.

Dermatology, 2000, 201(1), 15 - 20
Changes in keratin 6 and keratin 10 (co-)expression in lesional and symptomless skin of spreading psoriasis; Mommers JM et al.; BACKGROUND: Keratin 6 (K6) and keratin 10 (K10) are markers for epidermal hyperproliferation and differentiation, respectively, and are both expressed in the suprabasal layers of the epidermis . They may be co-expressed in different stages of the spreading psoriatic lesion, but single expression can also occur . OBJECTIVE: To investigate to what extent keratinocytes express K6 and K10, and to what extent they co-express K6 and K10 in different stages of the psoriatic lesion . We studied this in spreading psoriatic plaques . METHODS: Three 3-mm punch biopsies were obtained from the inner involved margin of a spreading lesion, from the uninvolved skin immediately adjacent to the spreading plaque, and from the distant uninvolved skin of 8 patients with incipient psoriasis . From 9 healthy volunteers, 3-mm punch biopsies were obtained as controls . After preparation of single cell suspensions of these biopsies, a triple staining protocol was performed with markers for K6 (monoclonal antibody LHK6B), K10 (monoclonal antibody RKSE60) and DNA content (TO-PRO-3 iodide) . Subsequently, cells were measured with a flow cytometer and the proportion of the markers was calculated using specific software . RESULTS: We observed a population of K6/K10-co-expressing cells, but also populations expressing only K6 . These subpopulations varied with the involvement of the lesion . There was a statistically significant difference between the inner margin and the outer margin with respect to the proportion of K6- and K10-expressing cells, whereas more K6-positive and K10-negative cells were detected in the inner margin of the lesions . The proportion of K6/K10-co-expressing cells in the inner margin was significantly different from the distant uninvolved skin . CONCLUSION: We confirmed that individual keratinocytes in psoriasis can express K6 or K10 depending on their localization in involved or uninvolved skin . There is a unique subpopulation of cells in the psoriatic plaques which co-express K6 and K10 . More studies are required to fully understand the pathogenic relevance of co-expression and single expression of K6 and K10 .

Folia Histochem Cytobiol, 2000, 38(3), 129 - 31
Encapsulation of parathyroid cells in hollow fibers: a preliminary report; Granicka LH et al.; The purpose of experiments was to evaluate the survival and functioning of human parathyroid cells after encapsulation in hollow fibers (HFs) . The polypropylene HFs K600(PP Accurel (Akzo-Nobel, Germany) of inner diameter 0.6 mm, wall thickness 0.2 mm, original or surface modified were used for encapsulation . Production of parathormone (PTH) by encapsulated cells was measured in vitro . HF were filled with parathyroid cell suspension and tightly closed . Encapsulated cells were cultured for 9 or 33 days in RPMI 1640 containing 10% FCS or in Chang's medium . The level of PTH, produced by encapsulated cells was evaluated in the culture medium with radioimmunoassay test (RIA) . The assays were performed every 2-4 days . The result of PTH assay was similar in both types of tested media as well as with unmodified and modified HFs, being 2-4 pg/ml of culture medium per 10(3) encapsulated cells . In conclusion, encapsulation in original or modified HFs ensures diffusion of nutrients from culture medium to encapsulated cells and allows for functioning of cells for at least 33 days in vitro.

Br J Ophthalmol, 2000 Sep, 84(9), 1004 - 7
Topical ophthalmic beta blockers may cause release of histamine through cytotoxic effects on inflammatory cells; van Beek LM et al.; AIM: To evaluate the effects of beta blockers used in ophthalmology on the release of histamine from mixed cell preparations containing human leucocytes and basophils . METHODS: A mixed leucocyte and basophil preparation was obtained from venous blood of healthy non-atopic volunteers . Cell preparations were then incubated with betaxolol, metipranolol, timolol, or carteolol . After incubation for 1 hour the histamine content of the supernatant was analysed by automated fluorometric analysis . Cell viability was tested by measuring lactate dehydrogenase (LDH) concentrations . RESULTS: Betaxolol and metipranolol in concentrations between 10(-2) M and 10(-3) M liberated histamine from human blood cells in a dose dependent manner . Carteolol and timolol had no effect on histamine at these concentrations . At the same concentrations LDH was also detected in the supernatants of cell suspensions incubated with metipranolol or betaxolol . CONCLUSIONS: Betaxolol and metipranolol induce substantial histamine release from human leucocytes, probably as a result of their cytotoxic effect.

Jpn J Cancer Res, 2000 Aug, 91(8), 853 - 8
The combined effect of electroporation and borocaptate in boron neutron capture therapy for murine solid tumors; Ono K et al.; 10 B-Enriched borocaptate (BSH) was administered intraperitoneally to SCCVII tumor-bearing C3H / He mice . Electroporation (EP) was conducted by using a tweezers-type electrode . The (10) B contents in tumors were measured by prompt gamma-ray spectrometry . The colony formation assay was applied to investigate the antitumor effects of boron neutron capture therapy (BNCT) and thereby to estimate the intratumor localization of BSH . The (10) B concentrations in tumors decreased with time following BSH administration, falling to 5.4(0 . 1) ppm at 3 h, whereas EP treatment (3 repetitions) 15 min after BSH injection delayed the clearance of BSH from tumors, and the (10) B level remained at 19.4(0.9) ppm at 3 h . The effect of BNCT increased with the (10) B concentration in tumors, and the combination with EP showed a remarkably large cell killing effect even at 3 h after BSH injection . The effect of BNCT, i.e., slope coefficient of the cell survival curve of tumors, without EP was proportional to tumor (10) B level (r = 0.982), and that of BSH-BNCT combined with EP lay close to the same correlation line . However, tumors subjected to EP after BSH injection did not show high radiosensitivity when irradiated after conversion to a single cell suspension by enzymatic digestion . This indicates that the increase of the BNCT effect by EP was a consequence of enclosure of BSH in the interstitial space of tumor tissue and not within tumor cells . This is different from a previous in vitro study . The combination of EP and BNCT may be clinically useful, if a procedure to limit EP to the tumor region becomes available or if an alternative similar method is employed.

Xenobiotica, 2000 Jul, 30(7), 665 - 81
Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes; Hewitt NJ et al.; 1 . The effect of cryopreservation on the metabolic capacity of monkey hepatocytes over 4 h in suspension and 24 h in culture was determined . Hepatocytes were diluted in a buffer containing 10% DMSO and frozen in a computer-controlled chamber . 2 . Initial ethoxyresorufin and ethoxycoumarin O-deethylase (ECOD) activities were the same in fresh and cryopreserved (CP) hepatocytes . ECOD activity in suspensions declined over 4 h but was the same in fresh and CP hepatocytes . 3 . The formation of testosterone hydroxy (OHT) metabolites (namely 6beta-OHT, 2beta-OHT, 16beta-OHT, 16alpha-OHT, 15beta-OHT, 2alpha-OHT and 6beta-OHT) was unaffected by cryopreservation . The loss of OHT activities over 4 h in CP and fresh whole cell suspensions was attributed to a loss of cofactor . CP hepatocyte cultures had equivalent OHT activities to freshly isolated hepatocytes . 4 . Initial UDP-glucuronyltransferase (UGT) activities, using the substrates 4-methylumbelliferone, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP whole hepatocytes . At later times, UGT activity was lower in CP than fresh hepatocytes but this was due to a loss of UDPGA . Initial sulphotransferase (SULT) activities, using the substrates 2-naphthol, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP hepatocytes . SULT activities were less stable than UGT activities but were the same in fresh and CP hepatocytes throughout the 4-h incubation . 5 . Initial glutathione S-transferase activities (using 1-chloro-2,4-dinitrobenzene) were the same in fresh and CP hepatocytes and both did not decrease over 4 h . 6 . CP monkey hepatocytes are a useful model for metabolic and cytotoxicity studies . These cells can be can be used either in suspension or in culture.

Radiat Res, 2000 Sep, 154(3), 301 - 6
Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis; Holl V et al.; The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood . As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro . BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy . Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days . Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide . It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons . Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2 . However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture . The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations . Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.

Cas Lek Cesk, 2000 May 24, 139(10), 305 - 8
{Determination of intracellular molecules in cells in hematopoietic malignancy using flow cytometry}; Touskova M et al.; BACKGROUND: Detection of membrane molecules by immunophenotypisation is a routine counterpart of the laboratory tests which are needed for the complex diagnostic procedures of hematopoetic malignancies at present . The immunochemical detection of intracellular molecules is essential in such cases, where the expression of surface molecules on malignant cells is poor, or where the results of other diagnostic analyses are unequivocal (cases with ectopic expression of other lineage-specific molecules and cases of biphenotypic leukemias) . METHODS AND RESULTS: We followed the procedure using 4% solution of paraformaldehyd in phosphate buffered saline for fixation of cells . Lysing solution G (Becton-Dickinson) was used for permeabilisation of cells to detect nuclear and cytoplasmatic molecules . The permeabilised cell suspension was than washed with 0.5% solution of Tween 20 in phosphate buffered saline . This simple and rapid procedure is readily available for the routine detection of intracellular molecules . This approach is important for the diagnosis of hematopoetic malignancies.

Anticancer Res, 2000 Jul-Aug, 20(4), 2773 - 8
Flow cytometric analysis of antigen expression in malignant and normal renal cells; Li G et al.; Flow cytometry allows quantitative analysis of cancer cells . The aim of this study was to make a quantitative study of antigen expression in malignant and normal renal cells in order to find the efficient monoclonal antibodies (mAbs) for labelling renal cancer cells . MATERIAL AND METHODS: 15 malignant and adjacent normal renal tissues and three renal carcinoma cell lines (ACHN, A704 and CAKI-2) were analyzed . The malignant and normal renal tissues were dissociated mechanically into cell suspension . The mAbs and isotype controls were used for immunochemical labelling . The stained cells were analyzed by flow cytometry . RESULTS: Renal tumor associated antigen G 250 was frequently detected in malignant renal cells but not in normal renal cells . Renal tumor associated antigen gp200 recognized by 66.4.C2 and PN-15 was frequently detected in malignant cells, normal renal cells and also in all three carcinoma cell lines . Epithelial antigens were strongly positive in normal renal cells . Compared with MOC 31, Ber-EP4 and E 29, W-lD9 was mostly reactive to malignant renal cells . VU-1D9 was strongly positive on ACHN and A704 . The carbohydrate carcinoma antigens CA 125, DF3 and Sialyl Lewis(a) were detectable in some of the malignant and normal renal cells . Sialyl Lewis(a) could be weakly detected on ACHN and A 704 . Pan-cytokeratins and cytokeratin (CK) 8 were strongly expressed in malignant and normal renal cells and in all three cell lines . CONCLUSION: Our results indicated that G 250, 66.4.Ca, PN-15, VU-1D9, MNF116 and anti-ckg were efficient mAbs for labelling renal cancer cells . Their potential clinical application by flow cytometry should be explored.

J Biomed Mater Res, 2000 Nov, 52(2), 346 - 53
Microfabricated elastomeric stencils for micropatterning cell cultures; Folch A et al.; Here we present an inexpensive method to fabricate microscopic cellular cultures, which does not require any surface modification of the substrate prior to cell seeding . The method utilizes a reusable elastomeric stencil (i.e., a membrane containing thru holes) which seals spontaneously against the surface . The stencil is applied to the cell-culture substrate before seeding . During seeding, the stencil prevents the substrate from being exposed to the cell suspension except on the hole areas . After cells are allowed to attach and the stencil is peeled off, cellular islands with a shape similar to the holes remain on the cell-culture substrate . This solvent-free method can be combined with a wide range of substrates (including biocompatible polymers, homogeneous or nonplanar surfaces, microelectronic chips, and gels), biomolecules, and virtually any adherent cell type .

J Biomed Mater Res, 2000 Nov, 52(2), 246 - 55
Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy; Ponticiello MS et al.; Adult mesenchymal stem cells (MSCs), found in the bone marrow, have the potential to differentiate into multiple connective tissue types, including cartilage . In this study, we examined the potential of a porous gelatin sponge, Gelfoam, for use as a delivery vehicle for MSCs in cartilage regeneration therapy . Adult human MSCs (hMSCs) were seeded throughout the gelatin sponge after a 2-h incubation period . When cultured for 21 days in vitro in a defined medium supplemented with 10 ng/mL of TGF-beta 3, hMSC/Gelfoam constructs produced a cartilage-like extracellular matrix containing sulfated glycosaminoglycans (s-GAGs) and type-II collagen, as evident upon histologic evaluation . Constructs loaded with a cell suspension of 12 x 10(6) cells/mL produced an extracellular matrix containing 21 microg of s-GAG/microg of DNA after 21 days of culture . This production was more efficient than constructs loaded at higher or lower cell densities, indicating that the initial seeding density influences the ability of cells to produce extracellular matrix . When implanted in an osteochondral defect in the rabbit femoral condyle, Gelfoam cylinders were observed to be very biocompatible, with no evidence of immune response or lymphocytic infiltration at the site . Based on these observations we conclude that Gelfoam resorbable gelatin sponge is a promising candidate as a carrier matrix for MSC-based cartilage regeneration therapies .

Br J Dermatol, 2000 Aug, 143(2), 307 - 12
A comparative study of Fas and Fas-ligand expression during melanoma progression; Soubrane C et al.; BACKGROUND: Impaired regulation of apoptosis is known to be associated with the development of various cancers . Fas receptor (APO-1/CD95) binding to its ligand, Fas-ligand (Fas-L), has been shown to trigger apoptosis in various cell types . OBJECTIVES: In this study, we examined CD95 and Fas-L expression on primary and metastatic melanoma cells from patients to investigate a potential correlation between these measures of apoptosis and different disease stages . PATIENTS AND METHODS: Primary melanoma cells were obtained after surgical resection from 19 patients and metastatic cells from fine-needle aspiration of lymph nodes or palpable subcutaneous lesions in 25 patients . Normal skin cells were obtained at skin biopsy of 10 healthy donors . RESULTS: Flow cytometric analysis revealed that CD95 and Fas-L expression was detected in all the kinds of cell studied . In whole cell suspensions, CD95 expression was significantly higher (P < 0.0001) in normal skin cells than in melanoma cells, whatever the stage studied . By contrast, we observed an increase in Fas-L expression in melanoma cells compared with normal ones . Subsequently, using a double staining method, we studied these measures on HMB45+ cells, a specific marker for melanoma cells, and found that CD95 expression was significantly higher (P = 0.0005) in primary than in metastatic cells while Fas-L expression was significantly increased (P = 0 . 0004) in metastatic compared with primary cells . Furthermore, a relationship was found between CD95 or Fas-L expression and Breslow thickness; as primary melanoma thickness progressively increased, the percentage of HMB45+ CD95+ cells decreased while that of HMB45+ Fas-L+ cells concurrently increased . CONCLUSIONS: These results suggest that downregulation of CD95 and upregulation of Fas-L in melanoma might be considered as concomitant with disease progression.

Lab Invest, 2000 Aug, 80(8), 1215 - 25
Culture of dendritic cells from a nonlymphoid organ, the thyroid gland: evidence for TNFalpha-dependent phenotypic changes of thyroid-derived dendritic cells; Croizet K et al.; Because they are sparsely distributed in tissues, dendritic cells (DC) present in nonlymphoid organs are difficult to isolate . Only DC from skin and lung have been successfully studied in culture . The objective of the present work was to investigate the possibility of isolating and culturing DC from an endocrine organ, the thyroid gland, which is particularly susceptible to the development of autoimmune processes . The study was conducted on pig thyroid glands to have sufficient amounts of starting material . This choice required the characterization of immunological reagents capable of recognizing DC markers in the pig species . Using a discontinuous trypsinization procedure, a DC population representing 2% to 3% of the thyroid cell suspension was reproducibly obtained . Isolated DC quantitatively attached to tissue culture-treated dishes and segregated from thyrocytes . DC identified as cells expressing major histocompatibility complex class II molecules, the mannose receptor, and the S100 protein were found to have a high capacity to internalize labeled ligands, dextran, and mannosylated albumin . These cells had a phenotype of immature DC . Secondarily, a fraction of DC detached from culture dishes, and floating DC had low or no endocytic activity, a characteristic of mature DC . Treatment of DC/thyrocytes cocultures with tumor necrosis factor alpha (TNFalpha) activated the transformation of immature DC into mature DC . These data show that DC isolated from the thyroid gland can be maintained immature or activated to undergo maturation in primary culture . The procedure of cell isolation and culture should be adaptable to human thyroid tissue for in vitro analyses of DC-mediated immune responses.

Exp Dermatol, 2000 Aug, 9(4), 266 - 70
Flow-cytometric characterization of normal versus psoriatic epidermis using improved cell separation methodology; Seegers BA et al.; Recently a new approach for epidermal cell characterization was developed: three-parameter flow cytometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin . The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin . Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions . The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin . The new methodology revealed data with a narrow range . In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation.

Exp Dermatol, 2000 Aug, 9(4), 240 - 7
Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro; Laihia JK et al.; It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation . Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function . In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo . To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion . The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR . The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later . The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen . HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo . Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro . We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.

Shock, 2000 Aug, 14(2), 144 - 9
Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma; Hasko G et al.; Polyamines are endogenous immunomodulatory molecules . Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide . In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions . Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages . The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected . In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production . The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice . The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase . In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody . In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10.

Int J Radiat Biol, 2000 Aug, 76(8), 1129 - 41
Exposure of human osteosarcoma and bone marrow cells to tumour-targeted alpha-particles and gamma-irradiation: analysis of cell survival and microdosimetry; Aurlien E et al.; PURPOSE: This study was designed to compare the cytotoxic effects of an alpha-emitting radioimmunoconjugate, which binds to osteosarcoma but not to bone marrow cells, with those of external gamma-irradiation . MATERIALS AND METHODS: The human osteosarcoma cell line, OHS-s1, and mononuclear cells from bone marrow (BM) harvested from healthy donors, were used for these experiments . Cells in suspension were added to various activity concentrations of the anti-osteosarcoma monoclonal antibody TP-3 radiolabelled with 211At . Following incubation for 1 h, unbound radioactivity was washed off and cell survival was determined from clonogenic assays . Microdosimetry was calculated based on binding and retention kinetics of 211At to the cells, as well as cellular and nuclear diameters . For comparison, cell suspensions were irradiated with a single dose of 60Co gamma-rays . RESULTS: 211At-labelled TP-3 showed heterogeneous binding to OHS-s1 cells, with a considerable variation among experiments . About 78% of the initially bound 211At decayed while associated with the OHS-s1 cells . D0 values estimated by microdosimetry were 0.33 (0.22-0.48, range) Gy and 1.18 (0.89-1.89) Gy for OHS-s1 and BM cells, respectively, whereas D0 values after external beam irradiation were 0.86+/-0.07Gy and 1.71+/-0.22Gy . The relative biological effectiveness (RBE) of 211At-labelled TP-3 at 37% survival was 3.43 for OHS-s1 and 1.55 for BM . CONCLUSIONS: High-LET targeted alpha-particle exposure killed osteosarcoma cells more effectively than bone marrow cells, although heterogeneous antigen expression among these tumour cells limited the magnitude of this effect.

Am J Clin Pathol, 2000 Aug, 114(2), 258 - 63
Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20; Cornfield DB et al.; The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic . We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments . Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining . CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression . In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH . This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH . The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question . Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined.

J Exp Bot, 2000 Apr, 51(345), 807 - 15
Limiting CO2 levels induce a blue light-dependent HCO3- uptake system in Monoraphidium braunii; Giraldez N et al.; The in situ photoactivation of an HCO3- uptake system in the green alga Monoraphidium braunii requires the irradiation of the cell suspensions with short wavelength radiation (blue, UVA and/or UVC) . Plasma membrane ATPase inhibitors block the uptake of this monovalent anion at pH 9 . M . braunii cells grown in high CO2 lack an HCO3- uptake system in their plasma membrane, but those grown in low CO2 can take up this anion at high rates . Cells grown in high CO2, transferred to CO2-limiting conditions in the light, start taking up HCO3- in 30 min, although they take 90 min to reach maximum rates of HCO3- transport . Therefore, this induction process seems to be triggered by low external CO2 concentration . In fact, increasing or decreasing the external HCO3- concentration does not induce the uptake system and only a decrease in CO2 concentration in the medium triggers the induction process . The appearance of the HCO3- transport activity is sensitive to cycloheximide, indicating that cytoplasmic protein biosynthesis is necessary for the induction of the uptake system . Photosynthetically active radiation, but not particularly blue light, is essential for induction of the uptake system to occur and the inhibition of photosynthesis by DCMU blocks it . From these results it can be inferred that when M . braunii cells detect a drop in CO2 concentration, they induce a blue light-dependent HCO3- uptake system.

J Exp Bot, 2000 Apr, 51(345), 685 - 93
Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture; Kawano T et al.; Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L . cv . Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration . Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS . The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction . The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected . Similar results were obtained using HRP . It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro . The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals . The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2 . In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions . The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.

J Biomed Opt, 2000 Apr, 5(2), 131 - 7
Light scattering from cells: the contribution of the nucleus and the effects of proliferative status; Mourant JR et al.; As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei . The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells . Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures . An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined . Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells . Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus . This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells . Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.

Fish Shellfish Immunol, 2000 Jan, 10(1), 21 - 31
Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures; Diago ML et al.; This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium . When TPS cultures were kept in the same flask, i.e . without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells . Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells . Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers . The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane . Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions . Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium . These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions.

Plant Sci, 2000 Jul 28, 156(2), 185 - 192
Comparison of the phytotoxic activity of the phytotoxin destruxin B and four natural analogs; Pedras MS et al.; A quantitative bioassay utilizing staining of plant cell suspension cultures of Sinapis alba was employed to establish a structure-phytotoxic activity correlation among destruxin B, homodestruxin B, and desmethyldestruxin B, toxins produced by Alternaria brassicae (Berk.) Sacc., the causative agent of Alternaria blackspot of brassicas . In addition, the phytotoxicity of destruxin B, homodestruxin B, and their respective metabolites hydroxydestruxin B and hydroxyhomodestruxin B were tested on resistant and susceptible plant species utilizing in planta leaf assays and leaf uptake of toxin solutions . Overall, the results obtained from punctured leaf and cell staining assays indicated that homodestruxin B (EC(50) 3x10(-4) M) was the most toxic of the five compounds, followed by destruxin B (EC(50) 5x10(-4) M), and desmethyldestruxin B (EC(50)&z.Gt;5x10(-4) M) . On the other hand, the hydroxylated destruxins (hydroxydestruxin B EC(50)&z.Gt;5x10(-4) M) were significantly less phytotoxic than the parent toxins.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 668 - 70
Nutrient medium optimization for rosmarinic acid production by Lavandula vera MM cell suspension; Pavlov AI et al.; The overall effect of NH(4)NO(3), KNO(3), and KH(2)PO(4) on the biosynthesis of rosmarinic acid and cell biomass by Lavandula vera MM cell suspension was studied by the method of the full factor experiment . Polynomial regression models were elaborated to give a quantitative description of the processes of biosynthesis of rosmarinic acid (Y(1)) and cell biomass (Y(2)) as a result of the variation of the concentration of NH(4)(+), 0.09 g/L < or = X(1) < or = 0 . 23 g/L; NO(3)(-), 2.44 g/L < or = X(2) < or = 3.02 g/L; and KH(2)PO(4), 0 . 170 < or = X(3) < or = 0.425 g/L . Optimization procedures according to the modified Simplex method allowed us to establish the optimal conditions for the biosynthesis of rosmarinic acid by Lavandula vera MM: X(1*) = 0.09 g/L; X(2*) = 3.02 g/L, and X(3*) = 0.170 g/L, where Y(1)max = 1786.74 mg/L (27 times higher compared with the cultivation in the standard Linsmayer-Skoog medium) . As a result, modified ingredients of the Linsmayer-Skoog nutrient medium were applied for the cultivation of Lavandula vera MM to achieve a maximum yield of rosmarinic acid.

Transplantation, 2000 Jul 27, 70(2), 327 - 35
Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations; Kung L et al.; BACKGROUND: The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency . Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency . In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA . METHODS: We compared their effects on NFATC2 dephosphorylation using Western analysis, interferon-gamma production using ELISA, and CN phosphatase activity using the CN assay in human peripheral blood leukocytes (PBL) and mouse spleen cell suspension . RESULTS: The FK concentration inhibiting 50% (IC50) of all three activities was approximately 0.2 microg/ml in human PBL, versus 5-20 microg/ml for CsA . Although inhibition of interferon-gamma secretion and NFATC2 dephosphorylation was complete, inhibition of CN phosphatase activity was incomplete with both drugs at saturation, particularly with FK . Inhibition of CN phosphatase activity was incomplete whether FK treatment was in vivo in mouse or in vitro in various human and mouse tissues, especially brain . Exogenous FKBP12 or CyPA increased CN phosphatase inhibition, suggesting that incomplete inhibition of CN phosphatase activity reflected limiting amounts of active immunophilin . CONCLUSIONS: These data contradict the prevailing assumption that immunophilins are abundant and not limiting for inhibition of CN by CsA or FK . Further, the observation that FK and CsA completely inhibit immune function without completely inhibiting CN suggests that the inhibition of immune function is not mediated by general CN inhibition but by inhibition of a subset of CN which is critical for lymphocyte activation.

Eur J Biochem, 2000 Aug, 267(16), 5005 - 13
Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture; Sasabe M et al.; We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans . INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment . INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively . To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses . The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases . These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.

Clin Exp Allergy, 2000 Aug, 30(8), 1166 - 71
Diagnosis of venom allergy by flow cytometry . Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release; Sainte-Laudy J et al.; BACKGROUND: Potent allergens such as hymenoptera venoms are capable of inducing severe and life threatening clinical reactions . Percentage of false negative results obtained by the usual diagnostical methods is comprised between 10 and 25% . OBJECTIVE: Evaluation of the sensitivity and the specificity of cellular tests and particularly evaluation of a new flow cytometric method . METHODS: Forty-five allergic patients having experienced a local, a systemic reaction or an anaphylactic shock and 10 controls having undergone hymenoptera stings without clinical reactions were selected on the basis of the clinical history, skin tests and specific IgE . Three cellular tests were performed on the same cell suspensions and in the presence of 2 ng/mL of rIL3: histamine release (RIA), leukotriene C4 release (ELISA) and basophil activation test (flow cytometry after double anti-IgE FITC, anti-CD63 PE labelling) . RESULTS: As compared to the clinical history, sensitivities of skin tests, specific IgE, flow cytometry, histamine release and leukotriene release were, respectively; 85%, 88%, 100%, 89% and 100% . Flow cytometric analysis of basophil activation showed a significant decrease of the mean fluorescence density and number of IgE positive cells and a significant increase of the number of CD63 positive cells . The 10 controls tested by flow cytometry were negative . CONCLUSION: As compared to the clinical history and to the other parameters tested here, flow cytometry showed a high sensitivity and a high specificity . The excellent correlation observed between this method and the other cellular tests such as histamine and leukotriene release are in favour of the specificity of flow cytomery and in favour of the use of this method for venom allergy diagnosis.

Magn Reson Imaging, 2000 Jul, 18(6), 689 - 95
Effects of cell volume fraction changes on apparent diffusion in human cells; Anderson AW et al.; Diffusion-weighted imaging was used to study the relationship between apparent diffusion coefficient (ADC) and cell volume fraction in cell suspensions and packed arrays . Cell volume fraction was varied by changing extracellular fluid osmolarity (for human glial cells) and by changing cell density (for human glial and red blood cells) . In packed arrays of glial cells, ADC increased 10% when cells shrank and decreased 13% when cells swelled . ADC decreased 34% as cell density increased from 0 to 72% . In erythrocyte suspensions, ADC decreased 90% as the cell density increased from 0 to 89% . These results agree with theoretical predictions.

Biochem J, 2000 Aug 15, 350 Pt 1, 215 - 8
Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals; Gieseg S et al.; This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species . U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups . In an incubation for 22 h, 1.2 mM peroxyl radicals was generated and each cell acquired 1.5x10(8) -OOH groups . These groups were located on the cell proteins; no lipid peroxidation was detected . The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals . There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins . The half-life of protein hydroperoxides in cell suspensions was approx . 4 h at 37 degrees C . Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species.

Cryobiology, 2000 Jun, 40(4), 360 - 9
Green fluorescent protein: A novel viability assay for cryobiological applications; Elliott G et al.; Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ . Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly . Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues . As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability . To the authors' knowledge, this is the first use of GFP in cryobiology applications . Intracellular GFP fluorescence was evaluated following slow freezing . Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath . Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr) . A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97 . A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay . Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions . The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate .

Gene Ther, 2000 Aug, 7(15), 1259 - 64
Assessing gene expression in vivo: magnetic resonance imaging and spectroscopy; Bell JD et al.; Recent developments in magnetic resonance imaging and spectroscopy afford the possibility of detecting and assessing transfer, expression and subsequent therapeutic changes of effector or marker transgenes noninvasively . In the field of MR imaging, 'smart' MR contrast agents are being developed, so called because they change their conformational structure and in so doing induce MR detectable changes in a given tissue . These agents become 'switched on' in response to physiological changes brought about by the enzymatic action of a given gene product (enzymes), and are being developed for use in intact cells, isolated organs and animal models . Ultimately, these agents hold the promise of bridging the gap between the laboratory and the patient with noninvasive detection of transgene expression in vivo in man . Similarly, magnetic resonance spectroscopy is being developed as a noninvasive method to assess transgene expression indirectly by means of MR visible intracellular markers . These markers take the form of intracellular endo/exogenous metabolites associated with exogenous enzyme expression and function . Again, this technique will be applicable to a variety of different situations, from cell suspensions through to clinical imaging of the whole body . In this article the unique opportunities for laboratory-based and clinical studies afforded by MR techniques are discussed.

Cytometry, 2000 Aug 1, 40(4), 327 - 35
Ultraviolet-induced detection of halogenated pyrimidines: simultaneous analysis of DNA replication and cellular markers; Hammers HJ et al.; BACKGROUND: We describe a new nonenzymatic methodology that allows the simultaneous detection of DNA replication and other cellular markers such as immunophenotyping . DNA replicating cells are identified by their incorporation of halogenated thymidine analogs, e.g., 5-bromo-deoxyuridine (BrdUrd) . METHODS: Irradiation with ultraviolet (UV)-B or UV-A light in the presence of Hoechst 33258 and subsequent treatment with a hypotonic buffer makes BrdUrd accessible to monoclonal antibodies (mAb), thus allowing its sensitive detection . RESULTS: The photolysis of BrdUrd in DNA with UV light is sequence dependent and results in DNA damage, allowing the detection of remaining BrdUrd using hypotonic conditions . However, treatment with other inducers of single or double- strand breaks of DNA such as gamma irradiation or hydrogen peroxide did not allow BrdUrd detection . The new methodology is compatible with both mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i.e., alcohols . The successful identification of CD34+, CD138+, or CD19+ cells out of heterogeneous cell suspensions and their cell-cycle analysis are described . Results correlated very well with acid denaturation (r = 0.972) . The average coefficient of variation (CV) of G(1) in the DNA histogram was smaller than 5%, resulting in good preservation of DNA distribution . Also, the signal-to-noise ratio was almost twice as high as for 2N acid denaturation, facilitating convenient discrimination of BrdUrd-positive cells . CONCLUSIONS: In contrast to previous approaches, this methodology eliminates the need for any additional enzymatic treatment such as DNA digestion or strand-break labeling after UV irradiation . The method is fast, convenient, and inexpensive and should be able to promote the use of halogenated pyrimidines in basic and clinical research of cancer, immunology, and pharmacology .

Cancer, 2000 Jul 15, 89(2), 288 - 96
Thymidylate synthase quantitation and in vitro chemosensitivity testing predicts responses and survival of patients with isolated nonresectable liver tumors receiving hepatic arterial infusion chemotherapy; Link KH et al.; BACKGROUND: Patients with isolated, nonresectable liver tumors may receive regional hepatic arterial infusion (HAI) chemotherapy with response rates of about 50% . The objective of this study was to investigate the value of thymidylate synthase (TS) determination in combination with in vitro chemosensitivity testing to predict the responses and survival of patients receiving HAI . METHODS: TS mRNA expression was quantitated using a reverse transcription-polymerase chain reaction technique with beta-actin as the internal standard . In vitro chemosensitivity testing was performed with tumor cell suspensions using the human tumor colony-forming assay (HTCA) . RESULTS: An analysis of the test combination in 24 consecutive patients revealed that 77% (10 of 13) of the sensitive and 9% (1 of 11) of the resistant patients had complete or partial clinical responses . Sensitive patients were 8.5-fold more likely to respond (P = 0.0036) and displayed with 32 months (range, 5-75 months) a longer median survival than resistant patients with 17 months (range, 3-28 months, P = 0.003) . Analysis of the Kaplan-Meier curves revealed that sensitive patients had a higher overall survival probability, as determined by the log rank test (P = 0.044) . CONCLUSIONS: These results suggest that the clinical outcomes of patients receiving HAI therapy may be predictable with TS quantitation and HTCA . It is possible, therefore, that this combination may be used in the future to select patients with liver tumors who will benefit from HAI before the start of regional chemotherapy .

Am J Physiol Heart Circ Physiol, 2000 Aug, 279(2), H657 - 71
Estimating oxygen transport resistance of the microvascular wall; Vadapalli A et al.; The problem of diffusion of O(2) across the endothelial surface in precapillary vessels and its utilization in the vascular wall remains unresolved . To establish a relationship between precapillary release of O(2) and vascular wall consumption, we estimated the intravascular flux of O(2) on the basis of published in vivo measurements . To interpret the data, we utilized a diffusion model of the vascular wall and computed possible physiological ranges for O(2) consumption . We found that many flux values were not consistent with the diffusion model . We estimated the mitochondrial-based maximum O(2) consumption of the vascular wall (M(mt)) and a possible contribution to O(2) consumption of nitric oxide production by endothelial cells (M(NO)) . Many values of O(2) consumption predicted from the diffusion model exceeded M(mt) + M(NO) . In contrast, reported values of O(2) consumption for endothelial and smooth muscle cell suspensions and vascular strips in vitro do not exceed M(mt) . We conjecture that most of the reported values of intravascular O(2) flux are overestimated, and the likely source is in the experimental estimates of convective O(2) transport at upstream and downstream points of unbranched vascular segments.

Planta, 2000 Jun, 211(1), 43 - 9
Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana; Huertas IE et al.; Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry . Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration . The magnitude of this release was dependent on the length of time the cells had been kept in the dark . Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells . External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport . Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) . The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark . Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3- . These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium.

Endocr Regul, 2000 Jun, 34(2), 73 - 81
The influence of thyroxine on intensity of energy metabolism in bone marrow myeloid cells and neutrophilic polymorphonuclear leukocytes of neonatal pig; Babych H et al.; OBJECTIVES: To investigate the participation of thyroxine in the regulation of energy metabolism in neutrophilic polymorphonuclear leukocytes and their bone marrow precursors . MATERIALS AND METHODS: The influence of L-thyroxine (T4; 4 mg/kg every 12 hr from day 2 to 10 of age) was estimated on the activity of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), NADP-dependent isocitrate dehydrogenase (ICDH) and cytochrome C-oxidase in bone marrow myeloid cells and circulating neutrophils of 3, 5 and 10 day (d) old piglets . Serum T4 and 3,5, 3'-triiodothyronine (T3) concentrations were estimated at every stage of experiment by radioimmunoassay . Bone marrow cells of myeloid lineage and blood neutrophilic polymorphonuclear leukocytes were separated by differential centrifugation of haematopoietic cell suspension using Ficoll-Hypaque gradients . RESULTS: The hyperthyroid status resulted in significant increase in PFK and LDH activity in myelokaryocytes of 3 and 3-5 d piglets, while the activity of HK and PK in the cells of 3-10 d animals remained unchanged . Moreover, ICDH activity in myelokaryocytes increased on day 10 and that of cytochrome C oxidase in bone marrow cells at all intervals . Marked increase in HK and LDH activity on day 3-5 was found also in blood polymorphonuclear granulocytes, while PFK and PK activity was increased during the whole period . At the same time even the increase in ICDH and cytochrome C-oxidase activity was observed, respectively, in 3 and 5-10 d old piglet neutrophils . Besides that, T4 inhibited G-6-PDH activity in myeloid cells on day 3 to 10 and did not influence the enzyme activity in circulating leukocytes . CONCLUSIONS: The administration of T4 resulted in preferential stimulation of oxidative stages of carbohydrate catabolism in myelocaryocytes, while the activity of glycolytic enzymes in these cells was less affected . On the contrary, the enzymes of glycolysis in blood neutrophils showed higher sensitivity to T4 action as compared to catalysts of oxidative reactions . The intensity of pentose phosphate pathway seems to be inhibited in bone marrow myelocaryocytes of T4 treated animals, while that in blood leukocytes remained unaffected.

Acta Physiol Scand, 2000 Jul, 169(3), 203 - 19
Ruled by waves? Intracellular and intercellular calcium signalling; Rottingen J et al.; The field of calcium signalling has evolved rapidly the last 20 years . Physiologists had worked with cytosolic Ca2+ as the coupler of excitation and contraction of muscles and as a secretory signal in exocrine glands and in the synapses of the brain for several decades before the discovery of cellular calcium as a second messenger . Development of powerful techniques for measuring the concentration of cytosolic free calcium ions in cell suspensions and later in single cells and even in different cellular compartments, has resulted in an upsurge in the knowledge of the cellular machinery involved in intracellular calcium signalling . However, the focus on intracellular mechanisms might have led this field of study away from physiology . During the last few years there is an increasing evidence for an important role of calcium also as an intercellular signal . Via gap junctions calcium is able to co-ordinate cell populations and even organs like the liver . Here we will give an overview of the general mechanisms of intracellular calcium signalling, and then review the recent data on intercellular calcium signals . A functional coupling of cells in different tissues and organs by the way of calcium might be an important mechanism for controlling and synchronizing physiological responses

Int Arch Allergy Immunol, 2000 Jul, 122(3), 216 - 23
Inhibitory effect of wheat germ agglutinin on mouse mast cell adhesion to fibronectin; Wyczolkowska J et al.; BACKGROUND: Mast cells play a critical role in allergic and inflammatory responses . The interactions between these cells and extracellular matrix components influence the distribution of mast cells in tissues and their biological responsiveness . It has been reported that the lectin wheat germ agglutinin (WGA) inhibits mast cell mediator release . We decided to investigate whether adhesion to fibronectin (FN), another mast cell function, which is upregulated following FcepsilonRI cross-linking, is also inhibited by WGA . METHODS: Mouse bone-marrow-derived mast cell line MCP5/L was used . For FcepsilonRI-dependent mast cell activation, MCP5/L cells were sensitized with mouse IgE antibodies . WGA was added to cell suspensions simultaneously with a challenging agent and, after an appropriate incubation period, beta-hexosaminidase release and adhesion to FN were determined . RESULTS: Both FcepsilonRI cross-linking-dependent mast cell adhesion to FN and mediator release were dose-dependently inhibited by WGA; however, the lectin concentrations required to induce maximum inhibition of adhesion were significantly lower . Furthermore, WGA inhibited phorbol-myristate-acetate- and A-23187-mediated mast cell adhesion to FN, i.e . processes that do not engage FcepsilonRI . The effect of WGA on FcepsilonRI-mediated secretion was reversed by GlcNAc . In contrast, combination of GlcNAc and NeuNAc or N, N'-diacetylchitobiose was required to reverse the inhibitory effect of WGA on mast cell adhesion . CONCLUSION: The characteristics of WGA-mediated inhibition of MCP5/L mast cell adhesion to FN suggest that mast cell integrins are targets of the inhibitory action of WGA and the sugar moieties on these receptors might be important for receptor function .

Biotechnol Bioeng, 2000 Sep 5, 69(5), 478 - 85
Hydrogen production by Anabaena variabilis PK84 under simulated outdoor conditions; Borodin VB et al.; Hydrogen production by autotrophic, vanadium-grown cells of Anabaena variabilis PK84, a cyanobacterial mutant impaired in the utilization of molecular hydrogen, has been studied under simulated outdoor conditions . The cyanobacterium was cultivated in an automated helical tubular photobioreactor (4.35 L) under air containing 2% CO(2), with alternating 12-h light (36 degrees C) and 12-h dark (14 degrees to 30 degrees C) periods . A . variabilis steadily produced H(2) directly in the photobioreactor during continuous cultivation for 2.5 months . The maximum H(2) production by the continuously aerated culture under light of 332 microE . s(-1) . m(-2) was 230 mL per 12-h light period per photobioreactor and was observed at a growth density corresponding to 3.6 to 4.6 microgram Chl a . mL(-1) (1.2 to 1.6 mg dry weight . mL(-1)) . Replacement of air with an argon atmosphere enhanced H(2) evolution by a factor of 2 . This stimulatory effect was caused mainly by N(2) deprivation in the cell suspension . A short-term decrease of the CO(2) concentration in the air suppressed H(2) evolution . Anoxygenic conditions over the dark periods had a negative effect on H(2) production . The peculiarity of hydrogen production and some physiological characteristics of A . variabilis PK84 during cultivation in the photobioreactor under a light-dark regime are investigated .

Tohoku J Exp Med, 2000 May, 191(1), 7 - 20
Auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration: short-term results; Abe T et al.; Autologous iris pigment epithelial cell transplantation was performed on patients with exudative age-related macular degeneration (AMD) . Autologous IPE cell culture was performed using autologous serum after iridectomy in 7 patients with AMD . The cell suspensions (2 approximately 20 x 10(4) cells) were transplanted into the submacular lesion of individuals after removal of neovascular membranes . Subsequent ophthalmological examinations, including best corrected visual acuity and fluorescein or indocyanine green angiography, were performed . In addition, 15 patients with AMD, who underwent removal of neovascular membrane without transplantation, were evaluated as non randomized controls . Varying degrees of atrophy or defects of choriocapillaris and retinal pigment epithelium were observed in all of the patients . No cystoid macular edema or fluorescein leakage was observed after treatment, but window defects were present . No patient had decreased visual acuity . One treated patient developed mild subretinal fibrosis and an other patient developed mild preretinal fibrosis, however no difference was significant when compared with the control . In conclusion, the treatment resulted in no significant improvement in macular function, as compared with the control; however, no rejection or deterioration in visual acuity occurred up to the 13 month follow up.

Magn Reson Med, 2000 Jul, 44(1), 144 - 56
NMR relaxation in tissues with weak magnetic inhomogeneities; Jensen JH et al.; A theory is presented for describing the effect on the transverse NMR relaxation rate of microscopic spatial inhomogeneities in the static magnetic field . The theory applies when the inhomogeneities are weak in magnitude and the nuclear spins diffuse a significant distance in comparison with a length scale characterizing the inhomogeneities . It is shown that the relaxation rate is determined by a temporal correlation function and depends quadratically on the magnitude of the inhomogeneities . For the case of unrestricted diffusion, a simple algebraic approximation for the temporal correlation function is derived . The theory is illustrated by applying it to a model of randomly distributed magnetized spheres . The theory is also used to fit experimental data for the dependence of the relaxation rate on the interecho time for a Carr-Purcell-Meiboom-Gill pulse sequence . The experimental systems considered are in vitro red blood cell suspensions and samples of human gray matter and rat liver . Magn Reson Med 44:144-156, 2000 .

Plant Physiol, 2000 Jul, 123(3), 1029 - 36
Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins; Inouhe M et al.; Suspension-cultured cells of azuki bean (Vigna angularis) as well as the original root tissues were hypersensitive to Cd (<10 microM) . Repeated subculturings with a sublethal level of Cd (1-10 microM) did not affect the subsequent response of cells to inhibitory levels of Cd (10-100 microM) . The azuki bean cells challenged to Cd did not contain phytochelatin (PC) peptides, unlike tomato (Lycopersicon esculentum) cells that have a substantial tolerance to Cd (>100 microM) . Both of the cell suspensions contained a similar level of reduced glutathione (GSH) when grown in the absence of Cd . Externally applied GSH to azuki bean cells recovered neither Cd tolerance nor PC synthesis of the cells . Furthermore, enzyme assays in vitro revealed that the protein extracts of azuki bean cells had no activity converting GSH to PCs, unlike tomato . These results suggest that azuki bean cells are lacking in the PC synthase activity per se, hence being Cd hypersensitive . We concluded that the PC synthase has an important role in Cd tolerance of suspension-cultured cells.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 99 - 102
{Effects of media on the production of flavonoids by suspension cultures of Saussurea medusa}; Zhao DX et al.; Flavonoids were produced from cell suspension cultures of Saussurea medusa . The results of studies on eight types of culture media showed that the MS medium was the best for cell growth and flavonoids formation, We investigated the effects of all the components of MS medium on the cell growth and flavonoids production, and found that carbon, nitrogen and phytohormone had especially marked effects . With MG medium a modified MS medium, the yield of cell growth was 24.8 g(dwt)/L, with MP medium another modified MS mediums, the yield of flavonoids production was 1 . 75 g/L . The yield of cell growth and flavonoids production in MG and MP medium were 32% and 70% higher than that in MS medium respectively.

Gen Comp Endocrinol, 2000 Jul, 119(1), 95 - 104
Effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail Coturnix coturnix japonica; Moore CB et al.; Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail . The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail . The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL) . In the second experiment, melatonin was administered in varying doses to adult quail placed in LL . The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 microg/ml melatonin given in the drinking water for 16 h per day for 2 weeks . The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment . In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response . To evaluate the humoral immune response, primary antibody titers were calculated 7 days postintravenous injection with a Chukar red blood cell suspension . In the adult birds of experiment 1, both the 8:16LD and 16:8LD treatments produced similar cellular and humoral immune responses but these responses were significantly greater than those observed in LL . The juvenile birds held under 8:16LD also had significantly greater cellular and humoral immune responses as compared to juvenile birds held in LL . In experiment 2, there was a clear melatonin dose response on immune function in LL . The humoral immune response increased to a peak at the 5.0 microg/ml dose while the cellular immune response increased across all dose levels . From the present study it was clear that quail placed in daily light-dark cycles (LD), possessing a diurnal rhythm of melatonin, had significantly elevated immune responses as compared to those birds in LL . Furthermore, melatonin supplemented to birds exposed to LL was immuno-enhancing . This suggests that melatonin may be a mediator of the differences seen between LD and LL lighting conditions and may have important immune modulating properties .

FEBS Lett, 2000 Jun 30, 476(1-2), 78 - 83
Chemical inhibitors: a tool for plant cell cycle studies; Planchais S et al.; Synchrony provides a large number of cells at defined points of the cell cycle . Highly synchronised cells are powerful and effective tools for molecular analyses and for studying the biochemical events of the cell cycle in plants . Usually, plant cell suspensions can be synchronised by chemical agents, which arrest the cell cycle by acting on the driving forces of the cell cycle engine such as cyclin-dependent kinase activity, enzymes involved in DNA synthesis or proteolysis of cell cycle regulators or by acting on the cell cycle apparatus (mitotic spindle) . The specificity, reversibility and efficiency of each type of cell cycle inhibitor are described and related to their mode of action.

Exp Neurol, 2000 Jul, 164(1), 209 - 14
Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum; Nishino H et al.; Epidermal growth factor (EGF)/fibroblast growth factor (FGF)-responsive stem (progenitor) cells from embryonic brain have self-renewing and multipotent properties and thus are good candidates for donor cells in neural transplantation . However, the survival and differentiation to mature neurons after grafting of stem cells into adult brain are rather poor . We hypothesize that the differentiation of stem cells to mature neurons, such as dopaminergic (DAergic) neurons, is dependent on environmental cues that control the ontogenic development . We compared the survival and differentiation between mesencephalic (MS) and cortical (CTx) stem (progenitor) cells, following grafting into bilateral striata of hemiparkinsonian model rats . MS and CTx stem cells were prepared from E12 rats and proliferated in serum-free medium with EGF or basic FGF for 2 weeks . One day after being primed to differentiate, the cell suspensions of both origins were grafted into the bilateral striata of adult rats that had unilateral 6-OHDA lesions in the substantia nigra . MS cells differentiated to tyrosine hydroxylase (TH)-positive neurons more strongly in DA-depleted striatum than in intact striatum, and methamphetamine-induced rotation was ameliorated in half of the grafted animals . Rosette-like cell aggregation and dysfunction of the blood-brain barrier (BBB) were less in and around the grafts in DA-depleted striatum, suggesting less proliferation and more differentiation of MS stem cells in DA-depleted striatum . Neither TH-positive neurons nor behavioral amelioration were detected following CTx stem (progenitor) cell transplantation in the striata . Data suggest that the DA-depleted striatum offers a suitable environment for MS stem (progenitor) cells to differentiate into mature DAergic neurons .

Exp Neurol, 2000 Jul, 164(1), 184 - 99
Long-term, EGF-stimulated cultures of attached GFAP-positive cells derived from the embryonic mouse lateral ganglionic eminence: in vitro and transplantation studies; Eriksson C et al.; Long-term attached cultures, prepared from mouse embryonic days 15-17 lateral ganglionic eminence, were grown in a medium including epidermal growth factor and serum, and the survival, differentiation, and migration of cells from either early or late passages were analyzed following transplantation . The cultured cells had the morphology of type I astroglial cells, with the vast majority of the cells immunoreactive for glial fibrillary acidic protein (around 90%), the intermediate filament marker nestin, and also the mouse-specific neural markers M2 and M6 . The cultures were kept over 25 passages (7 months) . During the first 8 passages, the growth rate gradually declined, but it increased again after passage 9 and thereafter stabilized at values similar to those observed during the initial culture period . After passages 4-6 and 18, cell suspensions were implanted cross-species into the intact or lesioned striatum of adult (passages 4-5 only) or intact striatum of neonatal rats (passages 4-6 or 18) . Both early and late passage cells formed M2 (and M6)-positive transplants . In the neonatal recipients, widespread migration was seen from the needle tract throughout most of the striatum, along the internal capsule, and into the globus pallidus . In the adult striatum, the cells remained mostly around the injection tract, or within 0.4-0.6 mm from the graft core . These long-term attached cultures are interesting to compare to nonattached neurosphere cultures, and might also offer a means of propagating relatively pure populations of astroglia-like cells for basic transplantation studies or for use in experimental trials with ex vivo gene transfer .

Exp Neurol, 2000 Jul, 164(1), 166 - 75
Failure of neuroprotection by embryonic striatal grafts in a double lesion rat model of striatonigral degeneration (multiple system atrophy); Puschban Z et al.; In the present experiment we studied the ability of embryonic striatal grafts to protect against striatal quinolinic acid (QA)-induced excitotoxicity in a previously established double lesion rat model of striatonigral degeneration (SND), the neuropathological substrate of parkinsonism associated with multiple system atrophy (MSA) . Male Wistar rats received under halothane inhalation anesthesia a 6-hydroxydopamine 6-OHDA injection into the left medial forebrain bundle . Four to 5 weeks later apomorphine-induced rotation behavior was tested . Rats were divided into two treatment groups receiving either embryonic striatal cell suspensions or sham injections . Apomorphine-induced rotation behavior was retested 2 and 4 weeks after the grafting procedure . Following the rotation test animals of the striatal and sham graft group received a stereotaxic injection of 150 nmol QA . Again rotation behavior was assessed 2 and 4 weeks after lesioning . Brains were then processed to dopamine reuptake ({(3)H}mazindol), dopamine D1 ({(3)H}SCH23390), and D2 ({(3)H}spiperone) receptor autoradiography . Gliosis was detected using {(3)H}PK11195, a marker for peripheral benzodiazepine binding sites . Behavioral and autoradiographic analysis failed to show striatal protection in 6-OHDA prelesioned animals receiving embryonic striatal grafts . These findings indicate that beneficial protective effects of striatal grafts implanted into host striatum prior to excitotoxic insults are abolished in the presence of severe dopaminergic denervation . Our present results are relevant to future applications of neural grafting in MSA-SND .

Exp Neurol, 2000 Jul, 164(1), 94 - 101
FK506 and cyclosporin A enhance the survival of cultured and grafted rat embryonic dopamine neurons; Castilho RF et al.; We examined the effects of the immunophilin ligands and calcineurin inhibitors FK506 and cyclosporin A on the survival of rat embryonic dopamine (tyrosine hydroxylase (TH)-immunoreactive) neurons . The protective effects of FK506 and cyclosporin A were first studied in dissociated mesencephalic cell cultures subjected to serum deprivation . Significant increases in both the total number of surviving mesencephalic cells and the number of surviving TH-immunoreactive neurons were observed when FK506 or cyclosporin A was present following withdrawal of serum from the culture medium . In a second series of experiments, FK506 increased the survival of dopamine neurons when added only to a hibernation medium in which donor tissue pieces were stored for 7 days prior to preparation of the cultures . In a third set of experiments, we investigated the effects of FK506 and cyclosporin A on the survival of grafted rat embryonic dopamine neurons . When FK506 or cyclosporin A was present during tissue preparation and in the final mesencephalic cell suspension used for grafting, the survival of TH-immunoreactive neurons implanted in the striatum increased to around 185% of control values . In contrast, treatment of graft recipient rats, but not the graft suspension itself, with immunosuppressive doses of FK506 or cyclosporin A did not augment the survival of grafted TH-immunoreactive neurons . We conclude that administration of FK506 during storage of embryonic mesencephalic tissue and FK506 or cyclosporin A during preparation of nigral cell suspensions used for grafting can increase the survival of grafted embryonic dopamine neurons .

Dig Dis Sci, 2000 Jun, 45(6), 1192 - 9
Use of flow cytometry to quantify mouse gastric epithelial cell populations; Zavros Y et al.; Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension . The quality of flow cytometry is strongly dependent on the isolation of intact viable cells . However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated . The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry . Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation . A Percoll density gradient was used to separate viable cells from cellular debris . Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry . According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%) . After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%) . Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively . T and B lymphocytes made up 4% and 2% in the gastric mucosa . Dissociated splenocytes were comprised of 20% T cells and 14% B cells . The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods . Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.

Brain Res Dev Brain Res, 2000 Jun 30, 121(2), 189 - 95
Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?
Crumpton TL, Seidler FJ, Slotkin TA.
The increasing use of chlorpyrifos (CPF) has elicited concern about neurotoxic effects on the fetus and neonate . CPF targets a number of events specific to brain development, over and above the ability of its active metabolite, CPF oxon, to inhibit cholinesterase . We used PC12 cells, a model system which displays many of the neurodevelopmental effects of CPF, in order to examine whether oxidative stress underlies the direct effects of CPF on development . Production of reactive oxygen species (ROS) was measured with a fluorescent intracellular dye . When PC12 cell suspensions were treated acutely with CPF for 10 min, ROS generation was increased in a concentration-dependent manner; CPF oxon was much less effective than the native compound . CPF also increased the ROS production in response to an acute sodium nitroprusside challenge, indicating sensitization of the cells to other oxidant stressors . Next, PC12 cells were grown in an undifferentiated state in the presence of CPF or CPF oxon for extended time periods, under conditions in which CPF inhibits mitosis, and the cells were then washed and ROS production measured . Neither compound elicited a significant change in ROS production . Finally, differentiation was initiated with nerve growth factor and the cells were exposed continuously to CPF or CPF oxon over a 72 h period; under these conditions, CPF inhibits neurite outgrowth . When the cells were washed and evaluated for ROS production, no significant differences were seen . These results indicate that CPF, but not CPF oxon, has the ability to elicit acute increases in ROS production . However, the effect disappears immediately once CPF exposure is terminated, possibly reflecting cellular defense mechanisms that lessen the impact of oxidant injury.

Phytochemistry, 2000 Jun, 54(3), 267 - 73
Taxonomic distribution of plant glutathione S-transferases acting on xenobiotics; Pflugmacher S et al.; Soluble and microsomal glutathione S-transferase activities for five model xenobiotics (nitrobenzene derivatives), two pesticidal xenobiotics (atrazine and fluorodifen), and a natural substrate (cinnamic acid), were determined in 59 different plant species and four plant cell suspension cultures . These enzyme activities were widely distributed over the plant kingdom with certain species showing particularly high activities . Marine macroalgae had a remarkably broad substrate range that included the substrates atrazine and fluorodifen . It is concluded that the evolutionary 'green liver' concept derived for xenobiotic metabolism in higher plant species is also valid for the constitutive soluble and microsomal glutathione S-transferases of lower plant species.

Vaccine, 2000 Aug 1, 18(28), 3319 - 25
Alkyl-polyacrylate esters are strong mucosal adjuvants; Hilgers LA et al.; Synthetic polymers were examined for their potency to enhance mucosal immune responses to inactivated antigens . Aqueous solutions of polyacrylic acid with a MW of 450 kDa (p{AA}) or an butyl-ester thereof with 16% esterification (Butyl16-p{AA}) plus antigen were administered twice intranasally in mice with a 2 week interval . The frequency of IgA-antibody secreting cells (ASCs) in lung cell suspensions was determined 1 week after the second immunisation . Both polymers significantly enhanced the IgA response against inactivated Newcastle disease virus (iNDV), inactivated influenza virus strain MRC-11 (iMRC-11), haemagglutinin/neuraminidase subunits of influenza virus strain A/Texas (HA/NA) and bovine serum albumin (BSA) . Butyl16-p(AA) was significantly more effective than non-derivatised p(AA), cholera toxin B subunit (CTB) or liposomes . The factor of increase in IgA-ASCs varied from <10- to >100-fold and depended on the type of antigen, the dose of antigen and the adjuvant . Extremely high responses of about 10,000 IgA-ASCs per million lung cells were detected after immunisation with 5 microg HA/NA plus 50 microg Butyl16-p(AA) . Intranasal immunisation with Butyl16-p(AA) resulted in high IgA responses, not only in the lungs, but also in the spleen and in high IgG responses in these organs . We concluded that alkyl-esters of polyacrylate are an interesting, novel category of mucosal adjuvants.

J Heart Lung Transplant, 2000 Jun, 19(6), 566 - 75
Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways; Stadlbauer TH et al.; BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models . We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days . The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection . METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection . At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides . RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours . Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard . Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion . Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days . In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment . CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients . Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.

Anal Cell Pathol, 1999, 19(3-4), 111 - 8
Hydrolysis profiles of formalin fixed paraffin-embedded tumors based on IOD (integrated optical density) and nuclear texture feature measurements; Flezar M et al.; The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin-embedded surgical samples, prepared as single cell suspensions for image cytometric measurements . The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature) . Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study . IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy . Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment . Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.

J Reprod Fertil, 2000 May, 119(1), 85 - 91
Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro; Hasthorpe S et al.; At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture . Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro . A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003) . Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies . The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01) . Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes . Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone . However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation . Evidence for the expression of the Mullerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive . This finding was in agreement with the lack of a direct action of Mullerian inhibiting substance on the formation of gonocyte-derived colonies in vitro . Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation . An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.

Mutat Res, 2000 Jun 22, 468(1), 73 - 85
In vivo DNA damage in gastric epithelial cells; Everett SM et al.; A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited . The comet assay is a simple technique for determining levels of DNA damage in individual cells . In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage . Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking . The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content . A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied . Cells were incubated with H(2)O(2) and DNA damage was assessed . Pronase and collagenase provided optimum digestion conditions, releasing 1 . 12x10(5) cells per biopsy, predominantly epithelial . Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20% . The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility . Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration . Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005) . Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt . Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt . The comet assay is a reproducible measure of DNA damage in gastric epithelial cells . Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.

J Immunol, 2000 Jul 1, 165(1), 404 - 10
Expression of P-selectin at low site density promotes selective attachment of eosinophils over neutrophils; Edwards BS et al.; The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail . We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer . We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils . Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below approximately 20 sites/microm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia . The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear . Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates . These differences may account in part for the increase in eosinophil accumulation in allergic diseases.

Anal Chem, 2000 Jun 1, 72(11), 2653 - 8
A screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; Aketani S et al.; A simple screening method is presented for the measurement of antigen-specific IgEs in sera in which mast cells are used . This method is based on the intracellular calcium signal in mast cells induced by cross-linking the surface high-affinity Fc receptors (FcepsilonRIs) with IgEs and multivalent antigens . When a serum containing various antigen-specific IgEs is added to the mast cell suspension, various antigen-specific IgEs are captured by FcepsilonRIs on the cell surface . However, the required antigen-specific IgE can be specifically detected after the addition of the corresponding antigen . The resulting increase in intracellular calcium concentration ({Ca2+}i), monitored by Ca2+-fluorometry, was found to be an analytical measure for the screening of IgEs . Two kinds of rodent mast cells, cell-lined RBL2H3 cells and primary cultured BMMCs, were used as a representative model system of mast cells . A DNP hapten (DNP35-HSA) and ovalbumin (OVA) were chosen for illustrative antigens, and these antigen-specific IgEs (DNP-specific IgE, OVA-specific IgE) in the corresponding rodent sera were target antibodies . It was found that {Ca2+}i increased linearly with IgE concentrations ranging from 25 to 5000 ng/mL for DNP-specific IgE and from 5 to 50 ng/mL for OVA-specific IgE . For these dynamic ranges, optimum concentrations of antigens were found to be 10 ng/mL and 1 microg/mL for DNP35-HSA and OVA, respectively . It was concluded that by monitoring the increase of {Ca2+}i in mast cells, we could determine the antigen-specific IgEs . The present immunological assay based on the Ca2+ signal transduction in mast cells offers new possibilities for efficient screening of antigen-specific IgEs and the immunogenicity of IgE in sera.

Zygote, 2000 May, 8(2), 97 - 105
Xenogeneic transplantation of human spermatogonia; Reis MM et al.; In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible . The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results . We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men . Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia . Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells . The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe . To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator . Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed . Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells . A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men . The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients . The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01) . The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes . A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection . However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found . The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

Photochem Photobiol, 2000 Jun, 71(6), 730 - 6
Autofluorescence patterns in short-term cultures of normal cervical tissue; Brookner CK et al.; Fluorescence spectroscopy has potential to improve cervical precancer detection . The relationship between tissue biochemistry and fluorescence is poorly understood . The goal of this study was to characterize normal cervical autofluorescence, using fresh tissue short-term tissue cultures and epithelial cell suspensions . Transverse, short-term tissue cultures were prepared from 31 cervical biopsies; autofluorescence images were obtained at 380 and 460 nm excitation . Fluorescence excitation-emission matrices were measured from normal, precancerous and cancerous cervical cell suspensions . Observed fluorescence patterns contrast those reported for frozen-thawed tissue, and were placed into groups with (1) bright epithelial and weak stromal fluorescence; (2) similar epithelial and stromal fluorescence; and (3) weak epithelial and bright stromal fluorescence . The average ages of women in the groups were 30.9, 38.0 and 49.2 years . Epithelial fluorescence intensity was similar in Groups 1 and 2, but weaker in Group 3 . Stromal intensity was similar in Groups 2 and 3, but weaker in Group 1 . The ratio of epithelial to stromal fluorescence intensity was significantly different for all groups . EEMs of cell suspensions showed peaks consistent with tryptophan, reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide . Short-term tissue cultures represent a novel, biologically appropriate model to understand cervical autofluorescence . Our results suggest a biological basis for the increased fluorescence seen in older, postmenopausal women.

J Neurochem, 2000 Jul, 75(1), 141 - 50
trans-resveratrol protects embryonic mesencephalic cells from tert-butyl hydroperoxide: electron paramagnetic resonance spin trapping evidence for a radical scavenging mechanism; Karlsson J et al.; In recent years, the antioxidant and other pharmacological properties of resveratrol, a natural product present in grapes and wine, have attracted considerable interest from the biomedical research community . In an examination of the potential neuroprotective properties of the compound, we have investigated the ability of resveratrol to protect rat embryonic mesencephalic tissue, rich in dopaminergic neurones, from the prooxidant tert-butyl hydroperoxide . Using the electron paramagnetic resonance (EPR) spin-trapping technique, the main radicals detected in cell suspensions were the tert-butoxyl radical and the methyl radical, indicating the one-electron reduction of the peroxide followed by a beta-scission reaction . The appearance of EPR signals from the trapped radicals preceded the onset of cytotoxicity, which was almost exclusively necrotic in nature . The inclusion of resveratrol in incubations resulted in the marked protection of cells from tert-butyl hydroperoxide . In parallel spin-trapping experiments, we were able to demonstrate the scavenging of radicals by resveratrol, which involved direct competition between resveratrol and the spin trap for reaction with the radicals . To our knowledge, this is the first example in which cytoprotection by resveratrol has been demonstrated by EPR spin-trapping competition kinetics to be due to its scavenging of the radicals responsible for the toxicity of a prooxidant.

J Virol, 2000 Jul, 74(13), 6087 - 95
Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells; Hu J et al.; Despite recent insights into mucosal human immunodeficiency virus (HIV) transmission, the route used by primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined . To determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the genital tract, rhesus macaques were intravaginally exposed to cell-free simian immunodeficiency virus SIVmac251 . We examined formalin-fixed tissues and HLA-DR(+)-enriched cell suspensions to identify the cells containing SIV RNA in the genital tract and draining lymph nodes within the first 24 h of infection . Using SIV-specific fluorescent in situ hybridization combined with immunofluorescent antibody labeling of lineage-specific cell markers, numerous SIV RNA(+) DC were documented in cell suspensions from the vaginal epithelium 18 h after vaginal inoculation . In addition, we determined the minimum time that the SIV inoculum must remain in contact with the genital mucosa for the virus to move from the vaginal lumen into the mucosa . We now show that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily intraepithelial DC and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal SIV exposure . The speed with which primate lentiviruses penetrate mucosal surfaces, infect DC, and disseminate to draining lymph nodes poses a serious challenge to HIV vaccine development.

Ann Transplant, 2000, 5(1), 14 - 20
Kinetics of bone marrow repopulation in lethally irradiated rats after transplantation of vascularized bone marrow in syngeneic hind limb; Lukomska B et al.; Grafting of the recipient with bone marrow cell suspension provides only few stromal cells and no autologous environment for cooperation between hemopoietic and stromal cells . Vascularized bone marrow grafts provide the recipient with bone marrow hemopoietic and stromal cells in their natural spatial relationship . It is expected that such a graft will resume its function soon after transplantation and almost immediately repopulate bone marrow cavities of the irradiated recipient as well as supply the sites of cytopoiesis with the necessary growth factors . The aim of the study was to investigate the process of repopulation of bone marrow cavities and lymphoid organs of irradiated recipient by bone marrow cells from syngeneic hind limb transplant . Lewis rats received total body irradiation of 8 Gy followed by orthotopic transplantation of syngeneic limb or i.v . infusion of equivalent amount of bone marrow cells . In control experiments the hind limb was shielded with lead plate during total body irradiation . Ten days after irradiation and hind limb transplantation the yield of nucleated cells from tibia was reaching values of normal animals . In rats receiving i.v . bone marrow cell infusion it was 40% of control values and in rats repopulated from shielded own hind limb it was 60% of controls . In all groups a higher percentage of early and immediate normoblasts and a reduced pool of juvenile and segmented neutrophils was observed . Thirty days after irradiation and repopulation procedures all parameters were returning to normal levels in each group . The results indicate that bone marrow cell transplantation in hind limb graft is highly effective in lethally irradiated animals in reconstituting bone marrow.

Immunol Cell Biol, 2000 Jun, 78(3), 294 - 6
Investigation of the site of precursors for IgA-producing cells in the chicken intestine; Muir WI et al.; Bursectomized chicks received lymphocyte single cell suspensions harvested from the bursa of Fabricius (BF), ileal lymphoid aggregate (ILA), caecal tonsils (CT), spleen and peripheral blood . Four days after cell transfer, repopulation of the duodenal and CT lamina propria in age-matched recipient bursectomized chickens with IgA-secreting plasma cells was determined . The results indicate the highest level of reconstitution with cells derived from BF, but substantial numbers of IgA-secreting plasma cells were also observed in a number of birds that received lymphocytes originating from the ILA and CT.

Phytochemistry, 2000 May, 54(1), 13 - 7
The configuration of methyl jasmonate affects paclitaxel and baccatin III production in Taxus cells; Yukimune Y et al.; All stereoisomers of methyl jasmonate (MJA) were prepared, and their effects on cell yield and promotion of paclitaxel (Taxol) and baccatin III production investigated in cell suspension cultures of Taxus media . (3R,7S)-MJA showed the strongest cell growth inhibition, followed by (3R,7R)-MJA . In contrast, (3S,7R)- and (3S,7S)-MJA had very low inhibitory effects, indicating that this inhibition depends largely on the (3R)-configuration . In terms of the promotion of paclitaxel and baccatin III production, (3R,7R)-MJA had the highest activity . Although it showed considerable activity at low concentration, at higher concentrations the activity was decreased due to strong inhibition of cell growth . Interestingly, paclitaxel and baccatin III contents increased even at a high (3S,7R)-MJA concentration, whereas the other isomers had the opposite effects . These findings are interpreted to suggest that the optimum configuration is (3R,7R), the (3R)-configuration not being indispensable, and that the (7R)-configuration is suitable for the promotion of paclitaxel and baccatin III production.

Dev Dyn, 2000 Jun, 218(2), 394 - 400
Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development; Grant TD et al.; Mice were generated in which a Col2-GFP transgene serves as a reporter for the chondrocyte lineage and for chondrogenesis in live embryos and newborn pups . Cells actively engaged in chondrogenesis were identified by confocal optical sectioning within their native environments in embryos and in thick tissue slices . Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed cell sorting of crude cell suspensions . Intensity of fluorescence correlated with biosynthesis of procollagen II in these cells . The use of these mice and their cells provides a novel approach for studying chondrocyte differentiation and chondrogenesis during skeletal development .

J Prosthet Dent, 2000 Jun, 83(6), 664 - 7
Effect of surface roughness of porcelain on adhesion of bacteria and their synthesizing glucans; Kawai K et al.; STATEMENT OF PROBLEM: In some instances of porcelain restoration, refinishing is inevitable . In terms of plaque accumulation on porcelain, refinishing could be a substitute method for glazing . PURPOSE: This study compared the amount of adhesion of plaque components (bacterial cells and glucans) on porcelain disks with various degrees of surface roughness to assess the effects of surface roughness on the amount of plaque accumulation . MATERIAL AND METHODS: Radiolabeled cell suspensions were incubated with porcelain disks for 3, 8, and 24 hours at 37 degrees C, and the amounts of adhered cells and glucans were measured by using a liquid scintillation method . RESULTS: The amount of cells and glucans adhered on porcelain increased with incubation time . The surface roughness value and the amount of plaque adhesion decreased with the increase in polishing level . However, the greatest amount of plaque was adhered on glazed surfaces, although their surfaces were smoother than the surfaces polished with 120- or 600-grit abrasive papers . CONCLUSION: With the exception of glazed surfaces, a positive correlation between surface roughness and the amount of plaque accumulation was observed . Repolishing with a diamond paste would not induce problems of plaque accumulation, compared with an intact glazed surface.

Haematologia (Budap), 2000, 30(1), 1 - 10
Thiorphan, an inhibitor of neutral endopeptidase/enkephalinase (CD10/CALLA) enhances cell proliferation in bone marrow cultures of patients with acute leukemia in remission; Stanovic S et al.; Thiorphan, (DL-mercapto-2-benzylpropanoyl)-glycine is a potent and specific inhibitor of membrane metallo-endopeptidase (EC 3.4.24.11, CD10) . We explored its effects in short-term clonal cultures of the bone marrow from 10 patients with acute leukemia in remission . The cell suspensions were incubated with thiorphan (10(-13) to 10(-5) M) and seeded for the granulocyte/macrophage-colony forming unit (GM-CFU) assay . In normal bone marrow samples the median seeding efficiency was 119 colonies and clusters per 10(5) cells and thiorphan caused slight stimulation of the clonal growth in concentrations above 10(-9) M . In the leukemic samples, the median seeding efficiency varied from 10 to 366 colonies and clusters per 10(5) seeded cells . Meaningful alterations of the clonal growth were noted in 32 out of 83 thiorphan-treated cultures (39%) . In those 32 cultures the stimulatory effects outnumbered the inhibitory effects (24 versus 8) . Thus, thiorphan stimulated the progenitor cell proliferation in bone marrow samples from the normal donor and from the patients with acute leukemia in remission . Thiorphan binding to CD10 might interfere with the processing of neuropeptide hemoregulatory factors and thus influence the progenitor cell proliferation.

Arch Toxicol, 2000 Apr, 74(2), 99 - 105
Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes; Nakagawa Y et al.; The relation between the metabolism and the cytotoxic effects of bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) has been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria . The incubation of hepatocytes with BPA (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by losses of intracellular ATP and total adenine nucleotide pools . BPA at a low-toxic level (0.25 mM) in the hepatocyte suspensions was rapidly converted to its major conjugate, BPA-glucuronide, and other minor products without marked loss of cell viability, although at a toxic level (0.5 mM), more than 65% of the compound presented in an unaltered form 2 h after the incubation . Addition of salicylamide (2 mM), non-toxic to hepatocytes during the incubation period, enhanced BPA-induced cytotoxicity and reduced the loss of BPA and the formation of BPA-glucuronide . The addition of BPA to isolated hepatic mitochondria caused a concentration (0-0.5 mM)-dependent increase in the rate of state 4 oxygen consumption in the presence of an FAD-linked substrate (succinate), indicating an uncoupling effect, whereas the rate of state 3 oxygen consumption was inhibited by BPA . Further, the addition of BPA (0.25 mM) reduced state 3 respiration with NAD+-linked substrates (pyruvate plus malate) and/or with the FAD-linked substrate, whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by BPA . A comparative study of the toxic effects of BPA and some bisphenols on cell viability (at 1.0 mM) and mitochondrial respiration (at 0.25 mM) revealed that 4,4'-(1,2-diethyl-1,2-ethenediyl)bisphenol (diethylstilbestrol) was more toxic than BPA, followed by 4,4'-methylenediphenol and 4,4'-biphenol . These results indicate that the onset of cytotoxicity caused by BPA may depend on the intracellular energy status and that mitochondria are important targets of the compound . The toxicity caused by the inhibition of ATP synthesis may be related to the concentration of unmetabolised free BPA remaining in the cell suspensions . In addition, the toxic potency of bisphenols to hepatocytes and mitochondria depends on the relative elongation and/or molecular size of the hydrocarbon bridge between the phenolic groups.

Prim, Care Update Ob Gyns . 1998 Jul 1, 5(4), 149 - 150
Potential for routine concurrent determination of chlamydia and cervical abnormalities by single fluid-based sampling; Lentrichia BB et al.; Objective: The FDA recently approved adjunctive HPV testing from the vial of PreservCyt(R) Solution used for the ThinPrep(R) Pap Test . We have now evaluated the potential for routine chlamydia testing from cellular material collected for primary Pap testing using the fluid-based ThinPrep method.Methods: Cervical scrapings were collected for a conventional Pap smear and residual material adhering to the sampling implement after slide preparation was suspended in PreservCyt Solution . Chlamydia testing was performed on this residual material using two methods . 1) A slide was prepared using the ThinPrep 2000 System for a determination by direct fluorescence . 2) One mL of the cell suspension was removed and processed for determination by an amplified DNA probe technique . A separate conventional endocervical swab sampling from the same patient was used for testing by an independent direct DNA probe technique (reference method) . One hundred specimens were selected and intentionally biased, 19 positive and 81 negative based on the independent swab results, for comparison to the slide and amplified DNA probe methods.Results: The slide-based method gave a sensitivity of 84% (16/19) and a specificity of 100% (81/81) . Of 3 discrepant specimens, 2 tested positive by the amplified probe method, while all 3 remained negative by repeat DFA . The amplified probe method gave a sensitivity of 95% (18/19) and a specificity of 99% (80/81) after analysis of discrepant samples by repeat testing.Conclusions: Feasibility has been demonstrated for accurate and effective routine testing for chlamydia with either DFA or DNA probe methods, from a single, fluid-based sample already approved for primary Pap screening.

FEBS Lett, 2000 Jun 2, 474(2-3), 217 - 22
Protein kinases induced by osmotic stresses and elicitor molecules in tobacco cell suspensions: two crossroad MAP kinases and one osmoregulation-specific protein kinase; Droillard MJ et al.; Two protein kinases displaying mitogen-activated protein kinase (MAPK) properties are activated both by an hypoosmotic stress and by oligogalacturonides in tobacco cell suspensions {Cazale et al . (1999) Plant J . 19, 297-307} . Using specific antibodies, they were identified as the salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) . The SIPK was also activated by an hyperosmotic stress, indicating that the same kinase may play a role both in hypo- and hyperosmotic signalling pathways, in addition to its involvement in the transduction of elicitor signals . Using immunoprecipitation followed by two-dimensional in-gel kinase assay, three molecular forms of the SIPK were observed, suggesting that additional modifications of the activated kinase may occur . In contrast to WIPK and SIPK, which are located at the crossroad of several transduction pathways initiated by elicitor or osmotic stimuli, a 44 kDa kinase, that would not belong to the MAPK family, appeared more specific to osmotic stress.

Int J Radiat Oncol Biol Phys, 2000 Jun 1, 47(3), 799 - 807
Combined effects of tirapazamine and mild hyperthermia on anti-angiogenic agent (TNP-470) treated tumors-reference to the effect on intratumor quiescent cells; Masunaga S et al.; PURPOSE: To evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470 . In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors . MATERIALS AND METHODS: C3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation . At the same time, the tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells . The mice then received TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT . Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point . After each treatment, 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 (or quiescent {Q} cells) was determined using immunofluorescence staining for BrdU . The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU . For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells . RESULTS: TPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells . In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice . Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment . CONCLUSION: Combined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.

Transplantation, 2000 Apr 27, 69(8), 1711 - 7
A comparison of fetal and adult porcine islets with regard to Gal alpha (1,3)Gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity; Bennet W et al.; BACKGROUND: It is still debated whether fetal or adult porcine islets should be the preferred choice for future clinical islet xenotransplantation . Each type of islet preparation has advantages and disadvantages compared with the other . Here we present a direct comparison between fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression, immunoglobulin and complement binding, and cytotoxicity after exposure to fresh human serum . METHOD: Islet single cell suspensions were prepared from adult and fetal islets by trypsin digestion . Fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (BS-IB4) and affinity-purified chicken anti-Gal alpha(1,3)Gal antibody was used to detect Gal alpha(1,3)Gal expression . Immunoglobulin and complement binding to the islet cells and cytotoxicity for islet cells was compared after incubation with fresh and heat-inactivated human sera and with an immune serum from a diabetic patient who received a fetal porcine islet transplant . Furthermore, two pools of human AB sera were depleted of porcine endothelial cell cytotoxic human anti-Gal alpha(1,3)Gal antibodies by absorption and were used to analyze the effect of Gal alpha(1,3)Gal antibody removal on islet cell cytotoxicity . RESULTS: Fetal islet cells readily bound both BS-IB4 and the chicken anti-Gal alpha(1,3)Gal antibody . None of 10 adult porcine islet preparations were stained by BS-IB4 . In comparison, IgY anti-Gal Ab binding was detected in two of eight adult islet isolations, whereas the other six preparations showed marginal/no binding . After incubation of fetal islet cells with fresh human serum, C3c binding was strongly positive and IgM binding variable, with occasional binding of IgG and no detectable binding of IgA . Adult islet cells were also strongly positive for C3c but did not bind detectable amounts of IgM, IgG, or IgA . Immune sera from a patient who had received fetal porcine islets showed the presence of induced antibodies that bound to fetal islet cells and to porcine peripheral blood lymphocytes, whereas binding to adult islet cells was barely detectable . Fresh human sera showed a high and similar level of complement-mediated lytic activity for both adult islet cells (78+/-22%) and fetal islet cells (75+/-16%) . Cytotoxicity for fetal islet cells and peripheral blood lymphocytes was significantly reduced when the corresponding sera were depleted of anti-Gal antibodies before use (P=0.002 and P=0.003, respectively) . In contrast, no difference in cytotoxicity for adult islet cells was detected when anti-Gal-depleted human sera were used . CONCLUSION: Gal alpha(1,3)Gal expression is occasionally detectable on adult porcine islet cells, but not as readily and at a lower level, compared with fetal islet cells . Thus, as porcine fetal islets mature to adult islets, the expression of the Gal alpha(1,3)Gal epitope gradually diminishes . Consequently, cytotoxic anti-Gal alpha(1,3)Gal antibodies in human serum play an important role in the lysis of fetal but not adult porcine islet cells.

Hum Pathol, 2000 May, 31(5), 584 - 92
A novel flow cytometric steroid hormone receptor assay for paraffin-embedded breast carcinomas: an objective quantification of the steroid hormone receptors and direct correlation to ploidy status and proliferative capacity in a single-tube assay; Leers MP et al.; Semiquantitative estimation of steroid hormone receptors by immunohistochemistry applied to paraffin sections is common practice in surgical pathology . Flow cytometric (FCM) analysis of estrogen receptor (ER) and progesterone receptor (PR) levels provides a faster and more objective quantitative assay . However, a major problem in such FCM analyses of solid tumor samples is the admixture of tumor cells with normal epithelial, stromal, and inflammatory cells . The aim of the underlying study was to investigate the applicability of a recently developed multiparameter flow cytometric methodology for the accurate estimation of the fraction of steroid hormone receptor-positive tumor cells and to explore whether this multiparameter approach allows the detection of specific, clinically relevant subsets of tumors, based on a combination of ploidy level, steroid hormone receptor status, and cell cycle characteristics . For this purpose, samples of 42 breast cancer patients, from which routine immunohistochemistry for ER and PR also was available, were analyzed . From each case, a cell suspension was prepared from the paraffin block by applying a heating and short pepsin digestion step to 50-microm-thick sections . These cell suspensions were double-immunostained for cytokeratin to identify the epithelial cells, and ER or PR, whereas DNA was quantitatively stained with propidium iodide using an optimized protocol . In the entire group of breast tumors, the percentages of ER- and PR-positive cells were registered in the epithelial subfraction, in combination with DNA ploidy and S phase fraction (SPF) . A significant correlation was found between the fraction of hormone receptor-positive cells as found by the immunohistochemical and FCM procedures . For ER, a correlation coefficient of r = .87 was found, and for PR r = .62, both P < .0001 . It became clear that all the diploid breast tumors had more than 30% tumor cells positive for ER with a SPF lower than 10%, whereas aneuploid tumors contained on average a smaller percentage of steroid hormone receptor-positive cells, and simultaneously an SPF greater than 10% . Our results show that this multiparameter FCM analysis allows an objective and reproducible quantification of the fraction of steroid hormone receptor-positive cells in the relevant epithelial cell compartment in relation to DNA ploidy status and proliferative capacity in a single-tube assay.

Jpn J Cancer Res, 2000 May, 91(5), 566 - 72
Usefulness of tirapazamine as a combined agent in chemoradiation and thermo-chemoradiation therapy at mild temperatures: reference to the effect on intratumor quiescent cells; Masunaga SI et al.; C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells . The mice then received one of six different DNA-damaging agents with or without mild temperature hyperthermia (40 degrees C, 30 min, MTH) . These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ) . After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of gamma-rays . Immediately 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 . The MN frequency in the total (P + Q) tumor cells was determined from the tumors that had not been pretreated with BrdU . MTH significantly increased the MN frequency of total cells in tumors irradiated with gamma-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with gamma-rays combined with BLM or TPZ . The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ . TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.

Biotechnol Prog, 2000 May-Jun, 16(3), 346 - 9
Production and elicitation of benzalacetone and the raspberry ketone in cell suspension cultures of Rubus idaeus; Pedapudi S et al.; Production levels of p-coumaric acid (p-CA), p-hydroxyphenylbut-3-ene-2-one (benzalacetone), and p-hydroxyphenyl-2-butanone (raspberry ketone) were measured in raspberry cell suspension cultures to investigate metabolite dynamics in a short (two-step) pathway . Intracellular concentrations of benzalacetone and the raspberry ketone fluctuated during the time course of a normal batch culture cycle but showed higher levels during periods of rapid growth . Cells elicited with the signal coupler methyl jasmonate yielded a 2- to 3-fold increase in metabolite concentrations after 24 h . The results suggest that raspberry ketone production is rapidly inducible during periods of high carbohydrate utilization . It is not an end product, however, and undergoes conversion to subsequent metabolites.

Dev Comp Immunol, 2000 Sep-Oct, 24(6-7), 663 - 72
Interferon-gamma induces differentiation of ectoplacental cone cells to phenotypically distinct trophoblasts; Athanassakis I et al.; Maturation of the murine ectoplacental cone results in the development of the placental tissue which essentially consists of two trophoblastic zones, the spongiotrophoblast and labyrinthine trophoblast . In this study we attempted to investigate the action of cytokines on ectoplacental cone cell differentiation to mature trophoblast cells . After determining the cellular composition of the ectoplacental cone cell suspensions based on the expression of cytokeratin, vimentin, Mac-1, class I and class II MHC antigens, the cells were exposed to the differentiation-inducing cytokines IL-3, GM-CSF, CSF-1 and IFN-gamma . From the four factors employed, only IFN-gamma increased the levels of cytokeratin-positive cells without inducing Mac-1 expression . IL-3 increased the percentages of cytokeratin as well as Mac-1- and vimentin-positive cells whereas GM-CSF and CSF-1 preferentially promoted an increase of the Mac-1 and vimentin markers . For further analysis, ectoplacental cone cells were negatively selected for Mac-1, class I and class II antigens to exclude non-trophoblastic contaminants and thereafter treated with the same cytokines . We show here that only IFN-gamma leads the sorted ectoplacental cone cells to a pure trophoblastic population composed of 100% cytokeratin-positive cells . The specificity of IFN-gamma on this differentiation pathway is strengthened by the fact that murine maternal serum on the day of EC formation contains high levels of this cytokine, suggesting that its natural presence supports - at least phenotypically - the in vivo differentiation of EC cells to trophoblasts.

Biosci Biotechnol Biochem, 2000 Apr, 64(4), 702 - 9
Teasterone-3-O-beta-D-glucopyranoside, a new conjugated brassinosteroid metabolite from lily cell suspension cultures and its identification in lily anthers; Soeno K et al.; The new brassinosteroid conjugate, teasterone-3-O-betaD-glucopyranoside, was found as a metabolite of teasterone in lily cell suspension cultures . Its structure was determined by means of FAB-MS and 1H-NMR upon comparison with the authentic compound . Furthermore, its presence in lily anthers was confirmed by FAB-MS and LC-APCI-SIM data . This is the first natural brassinosteroid conjugate glucosylated at a hydroxyl group in ring A.

Ultrasonics, 2000 Mar, 38(1-8), 638 - 41
Analytical scale ultrasonic standing wave manipulation of cells and microparticles; Coakley WT et al.; The ultrasonic standing-wave manipulation of suspended eukaryotic cells, bacteria and submicron latex or silica particles has been examined here . The different systems, involving plane or tubular ultrasonic transducers and a range of acoustic pathlengths, have been designed to treat suspension volumes of analytical scale i.e . 5 ml to 50 microliters for both sample batch and 'on-line' situations . Frequencies range from 1 to 12 MHz . The influence of secondary cell-cell interaction forces in determining the cell concentration dependence of harvesting efficiency in batch sedimentation systems is considered . Applications of standing wave radiation forces to (1) clarify cell suspensions, (2) enhance particle agglutination immunoassay detection of cells or cellular products and (3) examine and enhance cell-cell interactions in suspension are described.

Ultrasonics, 2000 Mar, 38(1-8), 633 - 7
Viability of yeast cells in well controlled propagating and standing ultrasonic plane waves; Radel S et al.; Recent studies have shown that there is no loss of cell viability when the cells are subjected to ultrasonic standing wave fields in acoustic cell retention systems . These systems are characterised by waves that spatially vary in pressure amplitude in the direction of sound propagation . In this work an anechoic 'one-dimensional' sonication chamber has been developed that produces propagating waves, which differ from standing waves in that the pressure amplitude remains constant as the wave travels in a medium with negligible attenuation . The viability of yeast cell suspensions as a function of treatment time was investigated during exposure to both standing and propagating wave fields with frequencies slightly above 2 MHz . The influence of 12% (vol/vol) of ethanol in water on the spatial arrangement of the cells in suspension was also studied . Changes in yeast cell morphology caused by the different types of suspension media and the ultrasonic treatment were examined by transmission electron microscopy (TEM) . The agglomeration of yeast cells within the pressure nodal planes appears to minimise damaging effects due to ultrasonic fields.

Ultrasonics, 2000 Mar, 38(1-8), 629 - 32
Viability of plant cell suspensions exposed to homogeneous ultrasonic fields of different energy density and wave type; Bohm H et al.; Exposure of Petunia hybrida cell suspensions to ultrasound at a frequency of 2.43 MHz in a standing wave field at an energy density of 70 Jm-3 (pressure amplitude of 0.78 MPa) decreased their mean viability to 35% after 20 min of sonication . A comparison of propagating wave and standing wave treatments at equal frequency (2.15 MHz) and energy density (8.5 Jm-3) showed, in the first case, a rapid decline in mean viability of cells (to 30% after 10 min of sonication) and, in the second case, a retaining of the initial viability (95%), respectively . Cells sonicated 4 days after subculture were more sensitive than cells sonicated 2 or 6 days after transfer to new culture medium . It was concluded that cellular viability depends primarily on the acoustic energy density, the exposure time, and the mechanical properties of the cells determined by age . As a consequence of the trapping of cells in the anti-node planes of the standing wave, propagating wave fields reduced cellular viability compared with standing wave fields at equal energy density.

Biophys J, 2000 Jun, 78(6), 3252 - 9
Visualization of myoglobin-facilitated mitochondrial O(2) delivery in a single isolated cardiomyocyte; Takahashi E et al.; The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats . Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution . Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation . Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered . Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions . Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells . Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.

Cancer Lett, 2000 Jul 31, 155(2), 153 - 61
Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential; Sato N et al.; We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer . The cells show rapid growth in culture with a doubling time of 16 h and high migration activity . Genetic and molecular analyses revealed high telomerase activity and a mutation in the K-ras oncogene . Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors . Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood . Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death . These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.

J Immunol Methods, 2000 May 26, 239(1-2), 13 - 23
Loss of CD34(+) hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents; Gratama JW et al.; Current protocols for sample preparation before flow cytometric enumeration of CD34(+) hematopoietic progenitor cells (HPC) include both lyse-non-wash and lyse and wash methods . Erythrocyte lysis without washing is the method of choice when absolute cell counts are to be assessed, whilst a washing step is recommended for immunological subtyping of CD34(+) cells in order to reduce background fluorescence . Here, we analyzed the effect of the interaction between type of erythrocyte lysis reagent and washing on the outcomes of (i) CD34(+) cell enumeration and (ii) expression of CD38 by CD34(+) cells in a single-platform, whole-blood staining assay {Gratama, J.W., Keeney, M., Sutherland, D.R., 1999 . Enumeration of CD34(+) hematopoietic stem cell and progenitor cells . Curr . Protocols Cytometry 6(4), 1-22.} . We studied seven commercially available lysing reagents (five containing fixative and two fixative-free) using 12 samples from cord blood (n=4), mobilized peripheral blood (n=4) and apheresis products (n=4) . Using the lyse and wash technique, significant reductions of absolute and relative numbers of CD34(+) cells, as well as in the numbers of lymphocytes and leukocytes, were observed on samples that had been lysed using fixative-containing buffers as compared to the lyse-no-wash technique . Cell losses due to washing could be significantly reduced when samples were lysed using fixative-free buffers . 'Postfixation' using PBS+1% paraformaldehyde of samples that had been lysed using fixative-free buffers and then washed did not result in additional loss of CD34(+) cells or other cell types . Finally, washing unfixed samples led to a slight decrease of CD38 monoclonal antibody bound to CD34(+) cells as compared to samples that had been fixed during erythrocyte lysis . These results indicate that fixation renders (CD34(+)) cells sticky and leads to their loss from the cell suspension upon centrifugation and resuspension . We conclude that all seven lysing reagents can be used with confidence in a lyse-no-wash technique, but that only fixative-free lysing reagents should be used when a washing step is considered necessary.

Hum Pathol, 2000 Apr, 31(4), 422 - 7
Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue: an additional diagnostic tool in surgical pathology; Leers MP et al.; A major drawback of immunohistochemical detection of monoclonality in B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining . To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used . Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material . We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10) . Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material . In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis . To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (kappa and lambda) was performed . This experiment showed that this cross-linking was minimal (less than 2%) . All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains . In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry . Four of the 9 cases were DNA aneuploid . One case did not show light chain expression at all by both techniques . However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-cell subpopulation . The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5% . The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1% . The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues.

J Surg Res, 2000 Jun 1, 91(1), 15 - 25
Amino acid uptake and regulation in multicellular hepatoma spheroids; Pawlik TM et al.; BACKGROUND: Cancer cells maintained in monolayer tissue culture are frequently used to study tumor biology and nutrient uptake, but there is a concern that this system may not fully reflect clinical tumor physiology . Because cells grown in a 3-D configuration more closely resemble an in vivo environment, a model was developed and characterized for the growth of SK-Hep human hepatoma cells in suspension as multicellular tumor spheroids (MTS) . The measurement of nutrient uptake in such a system has never been established . MATERIALS AND METHODS: SK-Hep cultures were initiated as single cell suspensions and grown as MTS in siliconized spinner flasks . The transport of several individual amino acids (arginine, glutamate, leucine, alpha-(N-methylamino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SK-Hep single cell suspensions and MTS (0 . 50-0.60 mm diameter) by a radiotracer/rapid filtration technique, as was the regulation of glutamine uptake by phorbol esters . l-{(3)H}GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter) . MTS cellularity was evaluated by histological examination, and single cell integrity after the transport assay was confirmed by scanning electron microscopy (SEM) . RESULTS: SK-Hep MTS displayed gradients of cellular morphology and staining, with central necrosis visible at diameters >0.8 mm . Single cell suspensions endured the rapid filtration technique based on functional Na(+)-dependent uptake rates and SEM analysis . Of all amino acids tested, only GLN transport rates were visibly affected by growth format . In small MTS, Na(+)-dependent GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm displaying central necrosis, when compared to single cell suspensions . Likewise, slight parallel changes in glutamine transporter ATB(0) mRNA levels were observed in Northern blot analysis . Finally, phorbol ester-dependent GLN transport down-regulation (by 40-50%), previously established in SK-Hep monolayers, remained operative in all cell formats tested . CONCLUSIONS: The data suggest that the tumor microenvironment differentially impacts the uptake of specific nutrients despite the conservation of key regulatory pathways . This MTS technique may prove useful for further studies on the role of nutrient transport in nascent tumor growth .

Biochem, Eng . J. . 2000 Jun 1, 5(2), 149 - 155
Releasing profiles of gene products from recombinant Escherichia coli in a high-voltage pulsed electric field; Ohshima T et al.; Release of recombinant proteins from gene-engineered Escherichia coli by applying a pulsed electric field (PEF) to a cell suspension was studied . When E . coli/pNC1, which produces beta-glucosidase and accumulates it in cytoplasm, was exposed to PEF, the most effective release of this enzyme was achieved in the cell suspension of 5% glycine and 15% PEG solution under 10kV/cm and 280J/ml of a PEF in a needle-plate electrode chamber . However, the amount of released beta-glucosidase by PEF treatment was only 26% of that by ultrasonic treatment . On the other hand, alpha-amylase produced by E . coli/pHI301A and accumulated in the periplasmic space could be easily released by PEF treatment . When this recombinant E . coli was suspended in 0.9% NaCl and 10% PEG solution and exposed to 10kV/cm and 200J/ml of a PEF in a plate-plate electrode chamber, 89% of intracellular alpha-amylase with nine-times higher specific activity compared with that by ultrasonic treatment was released . The release tendency of cellobiohydrolase, produced by E . coli/pNB6 and accumulated in both the cytoplasm and periplasmic space, was intermediate between those of beta-glucosidase and alpha-amylase . In this case, 70% of cellobiohydrolase with 1.9-times higher specific activity compared with that by ultrasonic treatment could be released when E . coli/pNB6 was suspended in 15% PEG and 10kV/cm and 200J/ml of a PEF was applied in a needle-plate electrode chamber . These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cytoplasmic membrane simultaneously . Therefore, PEF treatment is useful for easy release of periplasmic protein with selectivity.

Phytother Res, 2000 May, 14(3), 163 - 6
Ursolic acid isolated from Eucalyptus tereticornis protects against ethanol toxicity in isolated rat hepatocytes; Saraswat B et al.; Ursolic acid is the active material isolated from the leaves of the Eucalyptus hybrid E . tereticornis . In the present study, it has shown a significant preventive effect in vitro against ethanol-induced toxicity in isolated rat hepatocytes . Compared with the incubation of isolated hepatocytes with ethanol only, the simultaneous presence of ursolic acid in the cell suspension preserved the viability of hepatocytes and reversed the ethanol-induced loss in the level of all the marker enzymes (AST, ALT and AP) studied . Ethanol alone resulted in a 48%-54% decrease in the viability and a 42%-54% reduction in the biochemical parameters of the hepatocytes . Ursolic acid showed a concentration dependent (1-100 microg/mL) preventive effect (12%-76%) on alcohol-induced hepatocyte toxicity by restoring the altered parameters . The results thus suggest the effective use of an in vitro test system as an alternative for in vivo assessment of hepatoprotective activity of purified material .

Plant Sci, 2000 Jun 29, 155(2), 203 - 212
GFLV replication in electroporated grapevine protoplasts; Valat L et al.; Grapevine fanleaf virus (GFLV), responsible for the economically important court-noue disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes . We have developed direct inoculation of GFLV into grapevine through protoplast electroporation . Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions . Permeation conditions were determined by monitoring calcein uptake . Low salt poration medium was selected . Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13) . GFLV replication was detected with either virus particles (2 microg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting . Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate . These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

J Hematother Stem Cell Res, 2000 Apr, 9(2), 275 - 84
Competitive cytokeratin 19 RT-PCR for quantification of breast cancer cells in blood cell suspensions; Trummer A et al.; Detection of residual tumor cells in BM and PBPC products has been correlated with worse outcome of breast cancer patients . Still, there is a considerable demand for studies investigating the influence of the actual tumor cell number on prognosis, as quantification routinely has been cumbersome and time consuming and, thus, was evaded . We developed and evaluated a competitive RT-PCR-ELISA assay for cytokeratin 19 (CK19) with standard curve quantification that allows quantification of multiple samples within a working day; mRNA isolation, RT-PCR reaction, and automated ELISA detection were carried out using commercial kits . Results were expressed as OD420nm ratios of CK19 and an internal competitor . Values were then converted into tumor cell numbers using a standard curve of MCF-7 tumor cells . The assay had high specificity because of primers and capture probes with great heterogeneity to both published pseudogenes, which was confirmed by BLAST sequence alignment . We achieved a sensitivity of detecting 1 tumor cell per 10(6) mononuclear cells (MNC) . Between-batch precision (n = 8) for quantification was consistent and reasonable, with a coefficient of variation around 25% . Therefore, this assay should be suitable and sufficient for routine quantification of tumor cell numbers in BM or PBPC samples.

J Hematother Stem Cell Res, 2000 Apr, 9(2), 161 - 73
Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation; Woods EJ et al.; The transplantation of placental/cord blood-derived HPC (e.g., CD34+ cells) has become a useful treatment for a broad spectrum of malignant and nonmalignant diseases . The ability to cryopreserve this cell type with high efficiency adds considerable flexibility to cord blood transplantation . The purpose of this study was to develop an understanding of the fundamental cryobiologic factors of these cells, including the osmotic/permeability characteristics, and to use a theoretical approach to optimize freezing procedures . To that end, biophysical parameters, including the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), and cryoprotectant permeability coefficient (P(CPA)) for DMSO and propylene glycol were measured using a modified Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at 22 degrees C . In addition, the osmotic tolerance of PCB CD34+ cells was assessed using a colony-forming assay . These experimentally determined parameters were used in a mathematical model to predict optimal cryoprotectant addition and removal procedures . The results demonstrate a Vb of 0.32 x V(iso), an average Lp of 0.17 +/- 0.03 (microm/min/atm +/- SD), and a PCPA of 0.94 +/- 0.004 or 1.0 +/- 0.004 cm/min (x10(-3)) for DMSO or propylene glycol, respectively . No significant difference was determined between the two cryoprotectants used . The osmotic tolerance limits were determined to be 200 and 600 mOsm/kg (1.29 and 0.62 x V(iso), respectively) . These results indicate potential benefits of modifications to the widely used method of Rubinstein et al . Proc Natl Acad Sci USA 92:10119-10122, 1995) for cord blood CD34+ cell cryopreservation . As opposed to Rubinstein's method in which DMSO is added to cooled cell suspensions over a 15-min interval, our data indicate that better results may be obtained by introducing and removing the cryoprotectant at ambient temperature over 5 min both to increase viability by avoiding unnecessary risks from osmotic shock and to simplify the protocol . In addition, substitution of propylene glycol for DMSO may be of benefit during the actual freezing and thawing process.

Cell Transplant, 2000 Mar-Apr, 9(2), 153 - 68
Conditionally immortalized, multipotential and multifunctional neural stem cell lines as an approach to clinical transplantation; Gray JA et al.; Experiments are described using rats with two kinds of brain damage and consequent cognitive deficit (in the Morris water maze, three-door runway, and radial maze): 1) ischemic damage to the CA1 hippocampal cell field after four-vessel occlusion (4VO), and 2) damage to the forebrain cholinergic projection system by local injection of excitotoxins to the nuclei of origin or prolonged ethanol administration . Cell suspension grafts derived from primary fetal brain tissue display a stringent requirement for homotypical cell replacement in the 4VO model: cells from the embryonic day (E)18-19 CA1 hippocampal subfield, but not from CA3 or dentate gyrus or from E16 basal forebrain (cholinergic rich) led to recovery of cognitive function . After damage to the cholinergic system, conversely, recovery of function was seen with cell suspension grafts from E16 basal forebrain or cholinergic-rich E14 ventral mesencephalon, but not with implants of hippocampal tissue . These two models therefore provided a test of multifunctionality for a clonal line of conditionally immortalized neural stem cells, MHP36, derived from the E14 "immortomouse" hippocampal anlage . Implanted above the damaged CA1 cell field in 4VO-treated adult rats, these cells (multipotential in vitro) migrated to the damaged area, reconstituted the gross morphology of the CA1 pyramidal layer, took up both neuronal and glial phenotypes, and gave rise to cognitive recovery . Similar recovery of function and restoration of species-typical morphology was observed when MHP36 cells were implanted into marmosets with excitotoxic CAI damage . MHP36 implants led to recovery of cognitive function also in two experiments with rats with excitotoxic damage to the cholinergic system damage, either unilaterally in the nucleus basalis or bilaterally in both the nucleus basalis and the medial septal area . Thus, MHP36 cells are both multipotent (able to take up multiple cellular phenotypes) and multifunctional (able to repair diverse types of brain damage).

Tsitologiia, 2000, 42(3), 235 - 43
{Thermo- and radioinduced enzymatic relaxation of superhelical DNA from mud loach Misgurnus fossilis L . spermatozoa}; Nechaevskii IuV et al.; Actions of environmental impacts on mud loach spermatozoa were studied using various model systems: a) temperature stress, b) X-ray irradiation in vivo only of the animal head (a condition to trigger stress reaction), c) X-ray irradiation in vivo only of the animal body (a condition to exclude a direct activation of principal stress-realizing organism systems), d) gamma-irradiation in vitro of the cell suspension . It has been demonstrated that the temperature stress or X-ray irradiation of the mud loach head induced three lines of effects: 1) significant decrease in DNA superhelical density, 2) activity redistribution (functional activation) of DNase II between chromatin subfractions (with the increase of its association to chromatin), and 3) intracellular acidification up to pH value to satisfy the DNase II initiation . The obtained facts allow to suggest that, first, DNase II participates in the presented temperature- and radio-induced supercoiled DNA relaxation in spermatozoa, and, second, DNase II is involved in physiological (season elimination of spermatozoa that remained within male gonads after fertilization) or environmentally-induced DNA degradation.

Plant Sci, 2000 Feb 21, 151(2), 171 - 181
The seed-specific transactivator, ABI3, induces oleosin gene expression; Crowe AJ et al.; A microspore-derived cell suspension culture of Brassica napus was used as a host for expression studies involving seed oleosin genes . The suspension culture was previously shown to display biochemistry and gene expression typical of zygotic embryos . Using a biolistic, transient expression approach we demonstrate that the seed-specific activator ABI3 promotes oleosin gene expression in these cultures . Co-bombardment of an oleosin promoter-GUS fusion and a full-length ABI3 gene from Arabidopsis resulted in four to six-fold enhancement of GUS expression . Deletion analysis was performed to identify which oleosin upstream sequences were required for ABI3 regulation . These studies found that a truncated oleosin promoter containing 160 bp of 5' regulatory sequence was sufficient to confer ABI3 responsiveness . Mutation of a canonical abscisic acid response element within this 160 bp region had a dramatic effect on basal expression, reducing levels to 25% of control . However, this mutation had no significant effect on ABI3 transactivation, indicating that the reduction in basal oleosin expression was distinct from the ABI3 response . These results also suggest that ABI3-mediated transactivation occurs through either a less-conserved ABRE element or other abscisic acid-independent sequences within the minimal promoter . Together, these data provide the first direct evidence that ABI3 mediates oleosin transactivation.

AIDS, 2000 Apr 14, 14(6), 647 - 51
HIV-infected human Langerhans cells transmit infection to human lymphoid tissue ex vivo; Blauvelt A et al.; OBJECTIVES: To create a novel ex vivo model for early biologic events involved in sexual transmission of HIV and to demonstrate that Langerhans cells (LC), the purported initial mucosal target cells for HIV, play a critical role in this process . METHODS: Epidermal cells containing LC were isolated from normal-appearing skin of healthy volunteers and exposed to a panel of primary and laboratory-adapted R5- and X4-HIV isolates, washed and applied to the surfaces of allogeneic tonsil tissue blocks . Viral replication was followed by measuring HIV p24 protein in culture supernatants by ELISA . RESULTS: Both R5- and X4-HIV isolates could be transmitted by LC and established high levels of infection in lymphoid tissue (p24 > 10 ng/ml) . Depletion of LC within epidermal cell suspensions abrogated the ability of HIV-exposed suspensions to transmit virus to tonsil histocultures . CONCLUSIONS: Using a novel ex vivo model, human LC are shown for the first time to be the major epidermal cell type that is involved in transmission of HIV infection to human lymphoid tissue . Importantly, this system could prove useful in further understanding LC trafficking and other early biological events involved in primary HIV infection.

Lasers Surg Med, 2000, 26(4), 357 - 63
Laser modulation of angiogenic factor production by T-lymphocytes; Agaiby AD et al.; BACKGROUND AND OBJECTIVE: In previous investigations, small variations in the energy densities of low level light therapy (LLLT) were found to produce significant differences in the proliferation of resting T-lymphocytes in vitro . Pulsing these cells with mitogen in addition to laser therapy produced inhibitory effects regardless of the amplitude of the energy density used . In the current study, the effect of LLLT on the production of angiogenic factor(s) by T-lymphocytes was investigated in vitro . STUDY DESIGN/MATERIALS AND METHODS: Human T-cells isolated from peripheral blood were prepared in suspension either with or without addition of mitogen . Cell suspensions were irradiated with laser by using the following energy densities: 1.2, 3.6, 6.0, and 8.4 J/cm(2) . Wavelength, pulsing frequency, and power output were kept constant at 820 nm, 5,000 Hz, and 50 mW, respectively . After either 3 or 5 days of incubation, lymphocyte supernatants were collected and added as conditioned media to cultured endothelial cells (ECs) . The effect on the proliferation of these ECs was assessed over a 72-hour period by using a methylene blue assay . RESULTS: Endothelial cell proliferation increased significantly when incubated with conditioned media collected from resting T-cells exposed to 1.2 and 3.6 J/cm(2) . Day 5 conditioned media produced similar patterns of EC proliferation to that of day 3 but at lower magnitude . Pulsing of T-lymphocytes with mitogen in addition to laser irradiation significantly lessened their angiogenic capability . Conditioned media from 3.6 J/cm(2) laser-treated T-cells induced the maximal EC proliferation in all groups studied . CONCLUSION: It would seem that laser therapy stimulates lymphocytes to produce factor(s) that can modulate EC proliferation in vitro; this effect on the lymphocytes is influenced by (1) the amplitude of energy density used for T-cell irradiation, (2) exposing T-cells to both mitogen and laser, and (3) the duration of T-cell incubation in culture .

Oncol Res, 1999, 11(8), 367 - 73
Comparison of flow cytometry and RT-PCR for the detection of ovarian cancer cells in peripheral blood; Stimpfl M et al.; Recently, there has been significant effort in developing techniques designed to detect disseminated tumor cells in the peripheral blood (PB) . These techniques include immunocytochemical staining of cytocentrifuge slides, flow cytometry, and RT-PCR . Several authors reported various results concerning the sensitivity of the detection limit when applying these methods . The aim of this study was to assess the value of two methods in the detection of ovarian carcinoma cells in the PB . For tumor cell detection we compared RT-PCR to immunomagnetic enrichment followed by flow cytometric analysis . In a model system, single cell suspensions of ovarian cancer cell lines were mixed with full blood samples from healthy donors in order to determine the sensitivity limit of the two methods and to analyze the reproducibility of each . In a multiparameter flow cytometric analysis, tumor cells were defined as cytokeratin 7/8 positive and CD45 negative . RNA was screened for MUC1 mRNA by RT-PCR . MUC1 mRNA expression turned out not to be a specific marker of disseminated ovarian cancer cells, because a weak expression was also found in samples of healthy persons . Using immunomagnetic enrichment followed by flow cytometry, one carcinoma cell per 1 x 10(6) leukocytes was detectable . However, a minimum of 10 ml blood had to be analyzed in order to clearly distinguish real positive tumor cells from false-positive signals.

Toxicol Lett, 2000 May 19, 115(2), 153 - 63
Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells; Reiners JJ Jr et al.; Cultured cells are commonly exposed to trypsin-containing solutions in order to prepare cell suspensions suitable for subculture . Conditions used to release and disperse monolayers of cultured murine hepatoma 1c1c7 and human breast epithelial MCF10A cells caused the loss (40-95%) of cellular glutathione (GSH), but did not affect viability . Glutathione contents returned to pretrypsinization values within 24 h of replating . In contrast, the GSH contents of cultured rat hepatoma 5L cells were not affected by trypsinization . Exposure of 1c1c7 cultures to H(2)O(2) or etoposide 1 or 24 h after replating resulted in concentration-dependent cytostatic and cytotoxic effects . The concentration-response curves defining the cytostatic and cytotoxic effects of etoposide, and the cytostatic effects of H(2)O(2) were not influenced by the timing of toxicant addition . However, 1c1c7 cultures treated with H(2)O(2) 1 h after replating were more susceptible to the cytotoxic actions of the peroxide than cultures treated 24 h after plating . These studies show that conditions commonly used for the passaging of cultured cells can lead to a transient, but profound loss of GSH in some cell lines . Furthermore, the outcome of cytotoxicity analyses can be influenced by the time elapsed between the plating of cultures and the addition of toxicant.

Eur J Med Res, 2000 Apr 19, 5(4), 150 - 6
Expansion of CD60 helper lymphocytes detected in peripheral lymphocytes of HIV-1 infected individuals is not paralleled in lymph nodes; Bogner JR et al.; BACKGROUND AND OBJECTIVE: A significant expansion of CD8 cells with capability of Th2 type helper function had been observed in hemophiliacs with HIV infection . These cells were characterised by the surface co-expression of CD8 and CD60 antigen . Our objective was to investigate this lymphocyte subset in relation to other subsets in homosexuals and drug users in two compartments: blood and lymph nodes . Blood and lymph nodes from not HIV-infected persons served as control . RESULTS: CD8+CD60+ cells were expanded in perpheral blood of HIV - infected patients as compared to age matched controls (10.0 versus 4.1%, p <0.05) . This difference was not observed in lymph node cell suspensions (6.2 vs . 4.3% of all lymph node cells; p = n.s.) . The CD 4/CD8 ratio was significantly less impaired in lymph nodes than in blood (2.27 vs . 0.83; p <0.05) . Cytotoxic T cells were more abundant in the lymph nodes of patients with early stage HIV disease when compared two late stage patients (4.3 vs 2.1%; p <0.05) . Immunohistochemistry on frozen lymph node cuts showed presence of CD60 cells mainly in the interfollicular and paracortical area . In 3 of 10 HIV infected patients these cells were also found in the germinal centers . In controls no CD60 cells were detected in the follicles . Numbers and percentages of CD60 cellls and CD8+CD60+ cells in blood and in lymph nodes did not correlate with HIV - stage, CD4 count or plasma viral load . No correlation with lymph node viral load was seen . CONCLUSIONS: Our data confirm that like in hemophiliacs expansion of CD8+CD60+ is also found in the blood of other HIV risk groups and seems not to be specific for hemophiliacs . However, the higher percentage in peripheral blood is not paralleled in lymph nodes . Redistribution phenomena seem to be the most plausible explanation . According to these data, a major impact of CD8+CD60+ cells in the immunopathogenesis of HIV infection does not seem likely.

Invest Ophthalmol Vis Sci, 2000 May, 41(6), 1364 - 9
Telomerase activity and p53 expression in pterygia; Shimmura S et al.; PURPOSE: To investigate tolomerase activity and p53 expression in pterygial tissue . METHODS: Pterygia tissue was obtained during excisional surgery fr om 35 eyes of 35 patients, and superior bulbar conjunctival tissue from the same eye was also sampled as control when possible . Fluorescence telomeric repeat amplification protocol was used to measure telomerase activity in whole pterygium samples from 9 cases and in the epithelium and stroma of pterygium from another 10 cases . p53 protein content was measured by enzyme-linked immunosorbent assay (ELISA) in tissues obtained from 7 eyes, as well as in epithelial cell suspensions collected by brush cytology in 8 eyes . Six samples were also analyzed for UV-specific mutations in the p53 gene by the single-strand conformation polymorphism technique and DNA sequencing . A conjunctival epithelial cell line was irradiated with sublethal levels of UV-B to investigate whether telomerase activity can be induced in vitro . RESULTS: In all, 63% of pterygia samples demonstrated telomerase activity, whereas all 10 paired conjunctival control samples were negative (P = 0.05, chi-square test) . Of the 10 samples in which telomerase activity was measured separately in the epithelium and stroma of pterygia, 5 samples were positive in the epithelium, only 1 of which had activity in the stroma . Average telomerase activity in positive samples was 18.44 +/- 8.77 U/microg protein, compared with telomerase activity measured in a carcinoma in situ patient (33.73 U/microg), and in an immortalized conjunctival epithelial cell line (50.72 +/- 15.55 U/microg) . Telomerase activity was not upregulated in this cell line by UV-B exposure . All 6 pterygia samples tested for p53 mutations did not reveal the UV-specific mutations in exons 5, 6, 7, or 8 . No statistical significance was observed in the pterygium or conjunctiva p53 protein levels in epithelial cells collected by brush cytology, while p53 protein level was lower in pterygia when measured in whole tissue samples . CONCLUSIONS: Telomerase activity was detected in some pterygia, mostly in the epithelium . Pterygia was not associated with an increase in epithelial p53 protein content measured by ELISA.

Tissue Cell, 2000 Feb, 32(1), 66 - 70
Apoptosis of mouse embryonic stem cells induced by single cell suspension; Koyanagi-Katsuta R et al.; Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice . We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates . The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts . Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis . The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS . Cellular activity was restored by the addition of culture medium for ES cells . Glucose in the medium was found to be a major factor responsible for the restoration . Amino acids also restored the decrease in reduction of MTT . Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm . Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.

Plant Cell Physiol, 2000 Feb, 41(2), 244 - 50
Time-sequence observations of microtubule dynamics throughout mitosis in living cell suspensions of stable transgenic Arabidopsis--direct evidence for the origin of cortical microtubules at M/G1 interface; Hasezawa S et al.; Transgenic Arabidopsis thaliana, stably expressing a GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA . In the cell suspensions, the microtubular changes in the mitotic cells could be monitored by time-sequence observations using a time-lapse system of fluorescence microscopy . We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G2 phase to early G1 phase, and found that, at the M/G1 interface, the cortical MTs were firstly reorganized in the perinuclear regions and then in the cortex, as we had previously suggested (Hasezawa and Nagata 1991, Nagata et al . 1994) . The significance of this observation on the origin of cortical MTs is discussed.

Acta Ophthalmol Scand, 2000 Apr, 78(2), 122 - 9
Cyclosporine treatment of RPE allografts in the rabbit subretinal space; Crafoord S et al.; PURPOSE: To determine the effects of systemic cyclosporine A (CsA) on the survival of retinal pigment epithelial (RPE) allografts in the subretinal space in an animal model using atraumatic transplantation surgery . METHODS: Following pars plana vitrectomy, an RPE cell suspension from brown rabbits was injected with a glass micropipette into the subretinal space of 39 albino rabbits . For immunosuppression, 22 rabbits were given an injection of CsA, 20 mg daily intramuscularly, 17 rabbits with RPE grafts were controls . The grafts were monitored by biomicroscopy, color fundus photography, and fluorescein angiography . Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy including immunohistochemistry . RESULTS: After three months, the transplanted RPE cells, in both the CsA group and the controls, formed a monolayer in the subretinal space . Although a few macrophages were encountered, there was no massive cellular infiltration and the photoreceptor layer was well preserved . After six months, however, there was a disruption of grafted RPE cells in both groups, characterized by dispersion of melanin pigment in the subretinal space, and invasion of macrophages with focal photoreceptor damage but no infiltration of lymphocytes in the retina or choroid . No significant differences between the CsA treated and the control eyes were discernible . CONCLUSION: Although the subretinal space has been considered an immunologically privileged site, we found that the survival of RPE allografts was limited . CsA did not prevent RPE allograft destruction in the subretinal space . The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by nonimmunologic events.

Cancer, 2000 Apr 25, 90(2), 102 - 10
Adhesion of aspirated tumor cells to extracellular matrix proteins: a new methodology utilizing fine-needle aspiration; Gilcrease MZ et al.; BACKGROUND: Cell adhesion molecules mediate the interactions of cells with other cells and with extracellular matrix components . Such interactions may be important in the development of tumor invasion and metastasis . This article describes a new approach to the evaluation of tumor cell-matrix interactions by utilizing fine-needle aspiration of resected tumors . METHODS: Fine-needle aspiration was performed on 15 fresh surgical specimens of various types of carcinomas . After partial purification by isotonic Percoll centrifugation, tumor cell adhesion to collagen Type IV, laminin, and fibronectin was evaluated by counting cytologically malignant cells adhering to matrix-coated plastic substrates . Frozen tissue sections of the corresponding tumors were studied simultaneously for immunohistochemical expression of alpha-2, alpha-3, alpha-4, and alpha-5 integrin subunit expression . Results of the immunohistochemical staining then were compared with the adhesion data for particular tumors . RESULTS: In general, the majority of the tumors exhibited little or no adhesion to collagen or laminin, but several tumors showed marked adhesion to fibronectin . Striking differences were noted between some tumors of the same histologic subtype . Competitive inhibition studies performed with two of the tumors (a large cell carcinoma and a renal cell carcinoma) showed decreased adhesion to fibronectin in the presence of anti-alpha-5, suggesting at least a partial role for the alpha-5-beta 1 fibronectin receptor in mediating the adhesion of these tumors to fibronectin . All the tumors examined exhibited strong immunohistochemical expression of the alpha-2 and alpha-3 integrin subunits, and all were negative for alpha-4 . Three of the tumors showed weak expression of alpha-5, two of which (a squamous cell carcinoma and a renal cell carcinoma) were the tumors that showed the greatest adhesion to fibronectin . CONCLUSIONS: Quantitative adhesion data can be obtained using cell suspensions prepared from fine-needle aspirates, and there are marked differences in adhesive properties between particular tumors . Although two of the tumors showed a correlation between adhesion to fibronectin and immunohistochemical expression of the alpha-5 integrin subunit, matrix adhesion does not necessarily correlate with immunohistochemical expression of adhesion molecule receptors . In the future, this methodology potentially could be of value in determining which patients may benefit from therapies aimed at modifying tumor cell-matrix interactions.

Dev Biol Stand, 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, dimethyl-methylene 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.

Plant J, 2000 Apr, 22(2), 147 - 54
Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate; Munnik T et al.; In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses . In plant cells a signalling role for PLD and PA is also emerging . Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger . Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit . Increases occur within minutes of treatment and are dependent on the level of stress . Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32P-PA formation in a differential labelling protocol . Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell-suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum . These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.

Bioelectrochemistry, 2000 Feb, 51(1), 21 - 5
Attachment of bacterial cells to carbon electrodes; Morisaki H et al.; Anodic stripping method was applied to analyze the process of bacterial attachment to the surface of carbon-paste electrodes (CPE) . The electrode was immersed for various times in a bacterial cell suspension to allow the cells to attach to its surface . The number of bacterial cells attached to the electrode surface increased along with time . On the other hand, the current derived from the oxidation of a dye, Hoechst, which was adsorbed to the surface after attaching the bacterial cells, decreased along with time . It was considered that the current output, correlated with the amount of dye, adsorbed onto regions where no bacterial cell attached . These results indicate that the bacterial-attachment process can be analyzed by measuring the electric current derived from the dye instead of counting the number of attached cells.

Vet Q, 2000 Apr, 22(2), 117 - 20
Flow cytometric analysis of the DNA content of bovine and human bone marrow cells; Hoeben D et al.; The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow . Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry . Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration . Mononucleated cells were isolated by gradient centrifugation . In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase . The difference between calves and cows was not significant . In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase . A significant (P < 0.001) difference was observed between the two species . These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.

Leuk Res, 2000 May, 24(5), 427 - 35
Bone marrow purging by photodynamic treatment in children with acute leukemia: cytoprotective action of amifostine; Danilatou V et al.; In order to evaluate the combined effect of Amifostine and Merocyanine 540 during photoirradiation in neoplastic cells, bone marrow cells from children with acute leukemia (AL), age-matched controls as well as HL-60 cell line were studied . Cell suspensions were incubated with Amifostine, then with MC 540 and they were subsequently exposed to different irradiation doses by Argon Laser 514 nm . Cell survival was estimated by trypan blue supravital stain following a 24-h incubation . The leukemic cell line was studied in continuous liquid cell cultures for 4 weeks . The survival of normal bone marrow progenitors has been estimated by colony formation assay in methylcellulose cultures . Our results showed that Amifostine enhances the photokilling effect of MC 540 on leukemic cells and significantly protects bone marrow nucleated and committed progenitors (BFU-E and CFU-GM) from children with AL under chemotherapy . In conclusion, Amifostine seems to be a promising cytoprotective agent in the clinical use of purging with MC 540 mediated phototherapy.

Cell Transplant, 2000 Jan-Feb, 9(1), 45 - 53
Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor; Espejo M et al.; The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease . However, one objection to this procedure is the relatively poor survival of implanted cells . The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts . We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum . Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF . One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF . Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons . Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant . Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.

Physiol Res, 1999, 48(6), 417 - 27
Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes; Maletic SD et al.; Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO . Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN . ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner . This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05) . Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity . ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05) . In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis . Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished . A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production . The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN . In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes . These effects were mediated by NO as the effector molecule.

Hum Reprod, 2000 May, 15(5), 1125 - 35
The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing; Devireddy RV et al.; A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA) . Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution . Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA . Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume . By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined . The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2 . 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85 . 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg){cpa} = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp){cpa} = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98) . These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice . The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA . This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.

Lab Anim, 1999 Apr, 33(2), 175 - 84
The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial embolization of head and neck squamous cell carcinoma with dextran microspheres; van Es RJ et al.; A head and neck cancer model is developed using the VX2 carcinoma cell line injected s.c . in both ears of New Zealand White (NZW) rabbits . The study is focused on the effects of intraarterial embolization of the carcinomas with a new type of dextran hydrogel microspheres . During the phase of exponential growth the tumour-surface doubling-time was 7.1+/-2.0 days . Standard deviation in growth of the tumours was significantly larger between separate animals than between tumours growing in the left and right auricle of each individual animal (2.0 versus 0.65 days) . A fresh cell suspension containing at least 10 x 10(6) vital tumour cells was necessary to yield a tumour-take of 85% . The caudal auricular artery perfuses the caudal half of the external ear and is very suitable for macroscopic cannulation . Histological evaluation shows, that the use of dextran hydrogel microspheres of at least 25 microm in combination with ligation of non-tumour perfusing branches of the central auricular artery yields diffuse embolization of the VX2 carcinoma . This tumour model can be of use in further studies to optimize particle size and dosage for embolization as well as to evaluate the effect of different anti-neoplastic drugs, slowly released by controlled degradation of dextran microspheres.

J Chromatogr B Biomed Sci Appl, 2000 Jan 28, 738(1), 3 - 15
Simultaneous analysis of shikimate-derived secondary metabolites in Lithospermum erythrorhizon cell suspension cultures by high-performance liquid chromatography; Yamamoto H et al.; A high-performance liquid chromatography (HPLC) analysis system based on a water-acetonitrile gradient program was established for simultaneous quantification of shikimate-derived secondary metabolites in cultured cells of Lithospermum erythrorhizon . The cells cultured in pigment production medium (M-9) are capable of producing five highly hydrophilic compounds such as p-hydroxybenzoic acid-O-glucoside and lithospermic acid B, as well as eleven lipophilic compounds including echinofuran B and acetylshikonin . In addition to the wide polarities of those compounds, many of them are unstable under light, dryness, oxygen and heating . Thus, a new extraction procedure for all these compounds was also established by use of ultrasonication under ice-water chilling with MeOH as the solvent . This procedure was applied to the quantitative analyses of these compounds in cell cultures and hairy root cultures of Lithospermum, and in the intact plants as well.

Brain Res Brain Res Protoc, 2000 Apr, 5(2), 132 - 4
Measurement of intracellular calcium levels by the fluorescent Ca(2+) indicator Calcium-Green; Silei V et al.; The fluorescent calcium-sensitive indicators, such as the Calcium Green-1, allow one to detect small calcium transients at low indicator concentrations . The protocol reported here is a rapid and sensitive method that facilitates the measurement of intracellular free-calcium in cell suspensions . Using this assay, we were able to detect and quantify the variations in intracellular calcium concentration during microglial cell activation induced by the fragment peptides beta25-35 and PrP106-126.

Plant Sci, 2000 Jun 12, 155(1), 101 - 108
Cloning and characterization of eight cytochrome P450 cDNAs from chickpea (Cicer arietinum L.) cell suspension cultures; Overkamp S et al.; Eight different P450 sequences were isolated from a cDNA library derived from cultured chickpea cells (cultivar ILC3279) elicited with a Phytophthora sojae (formerly megasperma) elicitor (Pmg-elicitor) by screening with heterologous and homologous probes . Screening with CYP73A1 from Helianthus tuberosus yielded several clones with one identical sequence . A full-length clone could be isolated and this sequence was assigned CYP73A19 . Heterologous expression in yeast confirmed that CYP73A19 is the trans-cinnamic acid 4-hydroxylase of chickpea . Screening with a CYP81E2 polymerase chain reaction fragment from chickpea yielded a CYP81E2 full-length sequence and two almost identical CYP81E3 sequences, differing in only 16 out of 498 amino acids; both share more than 85% homology with the isoflavone 2'-hydroxylase from licorice (Glycyrrhiza echinata L.) . Using CYP93A1 as a probe, it was possible to isolate a full-length member of the CYP93 family, CYP93C3, that shares more than 80% homology with isoflavone synthase from soybean . In addition, partial sequences CYP81E3, CYP81E4 and CYP81E5 were also found in this screening . The use of a CYP82A2 probe derived from BAC F10N7 from Arabidopsis thaliana yielded only one sequence, CYP76F1 . Rescreening with CYP81E4 and CYP81E5 did not result in the isolation of any new P450 sequences . Northern blot experiments revealed that all but the CYP76F1 are induced rapidly and transiently in cell cultures upon elicitor treatment.

Anticancer Res, 2000 Jan-Feb, 20(1A), 21 - 6
Cell-surface expression of complement restriction factors and sialyl Lewis antigens in oral carcinoma: relevance to chemo-immunotherapy; Ravindranath NM et al.; Oral squamous cell carcinomas overexpress tumor-associated antigens, yet these antigens do not induce an immune-mediated anti-tumor response . The absence of an anti-tumor immune response may be due to poor immunogenicity of the tumor antigens or due to presence of factors that restrict immune functions . We have analyzed the expression of the tumor-associated sialyl LewisA (sLeA) and sialyl LewisX (sLeA) antigens, the complement restriction factors (CD59, CD46 and CD55) and the apoptosis associated factors Fas and Fas Ligand . Sialyl Lewis antigens (sLeA and sLeX), are immunogenic in that they elicit complement-fixing IgM antibodies . These antigens are associated with aggressive invasive behavior, tumor progression and poor disease-free survival of patients with human carcinomas . Human oral squamous carcinoma cell lines, SCC12 and SCC71, were analyzed for the density of Sialyl Lewis antigens, CD59, CD46, CD59, Fas and FasL on the cell surface . Expression of these antigens on the cell surface was determined employing a cell-suspension ELISA with monospecific monoclonal antibodies . In both oral carcinoma cell lines, the density of expression of sLeX was higher than that of sLeA and SCC71 had a very low level of sLeA expression . Both cell lines expressed a high density of CD59 and slightly lower levels of CD46 and CD55 on the cell surface, suggesting that even if host antibodies are accessible to the target antigens such as sLeX, they could not mediate complement-dependent cytotoxicity . The SCC lines expressed very low levels of Fas and FasL indicating that there maybe a lack of these signaling molecules for apoptosis . Our data suggests that passive immunotherapy or tumor killing by antibody-complement interaction may require downregulation of complement restriction factors.

J Exp Zool, 2000 May 1, 286(6), 632 - 40
ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture; Hirobe T et al.; It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro . Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes . In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions . Their potencies were compared with the potency of alpha-MSH . After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies . By 14 days, cultures contained mostly pigmented melanocytes . The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39) . The length of their peptide chains was inversely proportional to the potency . On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes . In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM) . These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely . The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.

J Biol Chem, 2000 Apr 21, 275(16), 11799 - 808
Transcriptional activation of the rice tungro bacilliform virus gene is critically dependent on an activator element located immediately upstream of the TATA box; He X et al.; To investigate the transcriptional mechanisms of rice tungro bacilliform virus, we have systematically analyzed an activator element located immediately upstream of the TATA box in the rice tungro bacilliform virus promoter and its cognate trans-acting factors . Using electrophoretic mobility shift assays, we showed that rice nuclear proteins bind to the activator element, forming multiple specific DNA-protein complexes via protein-protein interactions . Copper-phenanthroline footprinting and DNA methylation interference analysis indicated that multiple DNA-protein complexes share a common binding site located between positions -60 to -39, and the proteins contact the activator element in the major groove . DNA UV cross-linking assays further showed that two nuclear proteins (36 and 33 kDa), found in rice cell suspension and shoot nuclear extracts, and one (27 kDa), present in root nuclear extracts, bind to this activator element . In protoplasts derived from a rice (Oryza sativa) suspension culture, the activator element is a prerequisite for promoter activity and its function is critically dependent on its position relative to the TATA box . Thus, transcriptional activation may function via interactions with the basal transcriptional machinery, and we propose that this activation is mediated by protein-protein interactions in a position-dependent mechanism.

J Anim Sci, 2000 Mar, 78(3), 709 - 17
Suppressor activity of bone marrow cells and localization of fluorescent-labeled bone marrow cells within ovine endometrial tissue; Segerson EC et al.; Numbers of fluorescein isothiocyanate (FITC)-labeled bone marrow (BM) cells of donor lambs were quantified within endometrial cell suspensions following their administration to ovariectomized (OVX; control-and estradiol-17beta-treated) and intact (estrus, d-14 cyclic and pregnant) ewes . The numbers of fluorescent BM cells were greater (P < .05) for the estrous and d-14 cyclic ewes than for both groups of OVX ewes . Fractionation of the endometrial cells with Percoll revealed that the majority of fluorescent cells were low-density (1.002 to 1.056 g/mL) cells . In coculture experiments, low-density cells from lamb BM not only suppressed the incorporation of thymidine into phytohemagglutinin-treated peripheral blood lymphocytes, but the cells also released suppressor factor into the culture medium . Suppressor activity tended to be reversed (P < .1) by a pan-specific neutralization antibody to transforming growth factor-beta (TGF-beta); however, the activity was unaffected by a neutralization antibody to TGF-beta2 . These findings suggest that ovine endometrial suppressor cells may represent a population of low-density BM-derived natural suppressor cells, and their trafficking and localization patterns may depend on an ovarian factor(s) . Further, suppressor activity does not seem to be mediated by TGF-beta2.

Laryngorhinootologie, 2000 Mar, 79(3), 160 - 4
{Growth of human respiratory epithelium on collagen foil}; Bucheler M et al.; BACKGROUND: Clinical application of bioartificial tracheal prosthesis must still be regarded as an experimental concept because restoration of a functional respiratory epithelium outlining the prosthesis is still not possible . Tissue engineering as a relatively new biotechnological discipline may offer new methods in expanding differentiated respiratory epithelium in vitro . In this study we compare two different cell and tissue culture procedures for growing human nasal mucosa on commercially available collagen foil . MATERIAL AND METHODS: Harvested specimens of human nasal mucosa (n = 6, 4 x 4 cm) were placed on collagen foil and incubated as tissue cultures for 4, 6 and 8 weeks . A suspension of enzymatically dispersed nasal epithelium seeded on collagen foil (5 x 10(5) cells) served as control . Cell growth and ciliary beat were monitored through an inverted microscope with Hoffman's modulation contrast and video set-up . Histological examination was performed after 4, 6 and 8 weeks . RESULTS: In the tissue cultures, the collagen foil was initially covered with fibroblasts growing from the mucosa specimen before epithelial cells spread out . The epithelial layer showed mostly ciliated cells which developed metachronous ciliary beat after 4 weeks in vitro . Ciliary activity was observed until the end of the experiments in 8 weeks . New cells on the suspension cultures were mesenchymal and did not exhibit any ciliary activity . CONCLUSIONS: Mucosa specimens seem to be more appropriate for tissue engineering of respiratory epithelium than cell suspensions from nasal epithelium . Collagen foil as tissue scaffold initiates epithelial-mesenchymal interaction and may play an important role in epithelial differentiation of new respiratory epithelium.

Neurochem Res, 2000 Mar, 25(3), 357 - 62
In vivo indomethacin treatment causes microglial activation in adult mice; Prechel MM et al.; The current study was undertaken to study the role of prostaglandins in regulating microglial activation . Mice were treated with indomethacin (2 microg/ml) in their drinking water to selectively inhibit cyclooxygenase activity . After 4-8 days, the effect of inhibiting prostaglandin synthesis on microglial activity was evaluated . This was accomplished by analyzing microglial expression of Mac-1 (C3 complement receptor) as an indicator of activation . Mac-1 expression was assessed by immunohistochemistry of fixed brain cryosections, and by flow cytometric analysis of immunostained single cell suspensions . Both methods demonstrated that compared to age-matched, untreated controls, brains of indomethacin-treated mice had increased levels of Mac-1 expression, suggesting an increase in the state of microglial activation . These results demonstrate the importance of prostaglandins in down regulating microglial activity, and that inhibition of prostaglandin synthesis with indomethacin may act to increase the reactivity of the brain's immune system.

Cancer, 2000 Apr 15, 88(8), 1828 - 36
Gangliosides as targets for immunotherapy for pancreatic adenocarcinoma; Chu KU et al.; BACKGROUND: Pancreatic adenocarcinoma cells express gangliosides and sialyl Lewis (sLe) antigens . It is not known whether these carbohydrate antigens can be targeted by immunotherapy . The authors measured the expression of GM(2) and sLe antigens on the surface of pancreatic carcinoma cells and the serum levels of total gangliosides, GM(2), and antiganglioside antibodies in patients with pancreatic carcinoma . METHODS: Cell surface GM(2) and sLe antigens were measured by cell suspension enzyme linked immunoadsorbent assay (ELISA) in four pancreatic carcinoma cell lines . Sera from 20 pancreatic carcinoma patients and 20 age- and gender-matched healthy volunteers were analyzed for antiganglioside and anti-sLe immunoglobulin (Ig) M titers by ELISA . Serum levels of total gangliosides and GM(2) also were measured . RESULTS: All cell lines expressed GM(2) and sLe antigens . When compared with age- and gender-matched volunteers, patients had significantly higher serum levels of total gangliosides (25.6 +/- 9.0 mg/dL vs . 15.6 +/- 2.7 mg/dL; P < 0.001), GM(2) (0.278 +/- 0.415 mg/dL vs . 0.013 +/- 0.018 mg/dL; P = 0.02), ELISA units of anti-GM(2) IgM antibody (368 +/- 95 vs . 155 +/- 25; P = 0.04) and anti-GD(1b) IgM antibody (351 +/- 91 vs . 138 +/- 26; P = 0.03), but not anti-sLe(x) IgM (1389 +/- 345 vs . 1081 +/- 224; P = 0.46) or anti-sLe(a) IgM antibody (1097 +/- 253 vs . 1200 +/- 315; P = 0.80) . Patients with unresectable tumors had higher serum levels of total gangliosides compared with patients with resectable tumors, and a serum level > 25 mg/dL was found to correlate significantly with poor overall survival (P < 0.02) . CONCLUSIONS: Increased serum levels of total gangliosides and GM(2) may reflect shedding or release of gangliosides from the surface of tumor cells . Production of IgM antibody against GM(2) and GD(1b) indicates that these gangliosides are immunogenic antigens that may be potential targets for effective active immunotherapy .

Planta, 2000 Feb, 210(3), 478 - 87
Growth, ageing and death of a photoautotrophic plant cell culture; Peters W et al.; Batch cultures of photoautotrophic cell suspensions of Chenopodium rubrum L., growing in an inorganic medium on CO2 under a daily balanced light-dark regime of 16: 8 h could be maintained for approximately 100 d without subcultivation . The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3-4 weeks . These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division . Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins . Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells . However, responsiveness to the phytohormones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced . Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene . Similarities and differences between the development of batch-cultured photoautotrophic cells of C . rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf.

Planta, 2000 Feb, 210(3), 446 - 53
Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures; Mesnard F et al.; Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv . using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3 . By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected . Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline . Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components . A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N-acetylputrescine . The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored . For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.

Phytochemistry, 2000 Mar, 53(6), 659 - 65
Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins; Vitrac X et al.; We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L . The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy . In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar . We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation . Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production . Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose . The results with the two latter analogs are discussed . Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response . Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion . The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction . A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response . The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.

Phytochemistry, 2000 Mar, 53(6), 651 - 7
Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures; Yamamoto H et al.; Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin . The cells also produced a related dimer, (+)-rabdosiin . In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed . Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures . In the aerial parts of L . erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.

Anal Cell Pathol, 1999, 19(2), 81 - 90
Flow cytometric immunophenotyping of mature lymphatic neoplasias using knowledge guided cluster analysis; Barlage S et al.; Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies . Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi-color analysis is performed on highly heterogenous cell suspensions . We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non-Hodgkin lymphoma cells . The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population's location in two-dimensional dot plots . The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes . Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample.

Tohoku J Exp Med, 1999 Dec, 189(4), 295 - 305
Functional analysis after auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration; Abe T et al.; Recent transplantation studies indicate that subretinal space is not always an immunologically privileged site and non-autologous cells may be rejected in patients with exudative age-related macular degeneration (AMD) . We performed autologous iris pigment epithelial (IPE) cell transplantation by cell suspension after autologous IPE cell culture in 8 patients with AMD . These patients were followed without immunosuppression between 1.5 and 8 months and the retinal function was analyzed . No cystoid macular edema or fluorescein leakage was observed . Six of the 8 patients improved visual acuity of more than two lines and the other two patients retained preoperative visual acuity . Five patients had increased visual field sensitivity, one patient retained pretransplantation sensitivity, and one patient showed a gradual decrease in sensitivity (one patient was not examined) . Although 2 of the 8 patients showed decreased amplitude of flicker electroretinography (ERG) (about 60 to 70% as that of preoperative level), the average improvement of each amplitude of a single white flash (a wave), photopic, or flicker ERG was 123, 102, and 107%, respectively . No proliferative change in the submacular lesion or vitreous cavity was observed after transplantation . From this functional analysis, transplanted autologous IPE may have, in part, an alternative function in regard to the retinal pigment epithelium in the subretinal space.

Mol Reprod Dev, 2000 May, 56(1), 105 - 12
Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study; Golan R et al.; DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp . Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave . Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells . Acridine orange intercalated into double-stranded DNA to produce green fluorescence . The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix . When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases . The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin . The second phase presumably represents the period in which transition proteins are bound to the DNA . At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins . The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process .

Cell Biochem Biophys, 1999, 31(3), 231 - 45
Factors affecting yield and survival of cells when suspensions are subjected to centrifugation . Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields; Katkov II et al.; The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation . In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields . This paper mathematically analyzes the factors contributing to each goal . The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension . Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous . The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type . The behavior is modeled by the solution of coupled differential equations . The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

Protein Expr Purif, 2000 Apr, 18(3), 286 - 92
Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells; Karlsson M et al.; Monoglyceride lipase (MGL) has been produced with the baculovirus-insect cell system . The mouse MGL cDNA was subcloned into a baculovirus transfer vector in frame with a sequence encoding an N-terminal stretch of six histidine residues . Purification to apparent homogeneity was obtained by nickel-chelating chromatography . The final yield was 3 mg of pure enzymatically active MGL per liter of Sf9 cell suspension culture . Analysis by SDS-PAGE and mass spectrometry showed that the recombinant histidine-tagged enzyme had the expected molecular mass . With monoolein as substrate, the specific activity and the apparent K(m) were close to those of rat MGL of adipose tissue .

Theriogenology, 1998 Aug, 50(3), 465 - 77
Hormonal and immunological changes in blood and mammary secretion in the sow at parturition; Osterlundh I et al.; The purpose of the present study was to record possible variations of estradiol-17 beta (E2) and cortisol concentrations, and parameters related to granulocyte phagocytosis in mammary secretions from healthy sows at parturition . The study was comprised 8 primiparous sows (Landrace x Yorkshire) . Blood and mammary secretion samples were collected twice daily from 3 d before (only blood) until 3 d after farrowing . Estradiol-17 beta and cortisol concentrations were determined in plasma and in cell-depleted skimmed mammary secretions . Phagocytic capacity of polymorphonuclear cells (PMN) was assessed in whole blood and in cell suspensions derived from mammary secretions . Opsonic activity was assessed in serum and in cell-depleted skimmed mammary secretions . The 2 assays were based on chemiluminescence . Estradiol-17 beta concentration in plasma decreased (P < 0.001) directly after parturition . In skimmed secretions, the highest E2 concentration was recorded in the first sample after parturition and decreased (P < 0.01) thereafter . The highest cortisol concentration in plasma was recorded in the evening before parturition (P < 0.01) . In skimmed secretions, there was no significant variation in cortisol concentration . The concentrations of both steroid hormones were lower in mammary secretions than in plasma . The phagocytic capacity of PMN in blood and mammary secretion, expressed as peak chemiluminescence per PMN, showed no significant change . This was also true for the opsonic activity in serum . In skimmed secretions the opsonic activity increased (P < 0.01) after parturition . These data emphasize the differences between plasma and mammary secretion concentrations of steroid hormones as well as between systemic and mammary gland immune competence . Regarding the phagocytosis process in mammary secretions, the part directly related to the PMN function seemed not to be altered at parturition compared with later on in lactation, whereas the part related to opsonic activity seemed to be impaired at parturition . The latter may play a role in the development of coliform mastitis at this time.

Phytochemistry, 2000 Feb, 53(4), 447 - 50
Biotransformation of the Trichoderma metabolite 6-n-pentyl-2H-pyran-2-one by cell suspension cultures of Pinus radiata; Cooney JM et al.; Cell suspension cultures of Pinus radiata metabolize the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) (1) via hydroxylation of the pentyl side chain . Examination of the culture medium following dosing studies with 1 revealed that 79-85% of this bioactive compound had been metabolised after 144 h . At that time, 34-40% of the metabolized dose was recovered as a series of monohydroxylated isomers of 1, the principal metabolite being 5-(2-pyron-6-yl)pentan-5-ol (7).

Ital J Gastroenterol Hepatol, 1999 Nov, 31(8), 663 - 8
Comparison between McCoy cell line and HeLa cell line for detecting Helicobacter pylori cytotoxicity: clinical and pathological relevance; Xia HH et al.; BACKGROUND: Cell culture assay is an accurate test for detecting Helicobacter pylori cytotoxicity . AIMS: To evaluate McCoy cells for detecting Helicobacter pylori cytotoxicity by comparing with HeLa cells, and determine the association of cytotoxic strains with endoscopic and histological findings . METHODS: Helicobacter pylori isolates from 68 dyspeptic patients and 11 asymptomatic volunteers were tested . 180 microl (1.8 x 10(4) cells) of grown McCoy or HeLa cell suspension was seeded into each well of a 96-well microtitre tray and the medium was replaced once after 24 hours . Sonicate (20 microl) of each isolate was then added to the wells, in duplicate . After 24 and 48 hours incubation, intracellular vacuolation was assessed by inverted light microscopy . RESULTS: Using McCoy cells 57% of isolates showed cytotoxicity (23% weak and 34% strong), while using HeLa cells 30% of isolates showed strong cytotoxicity . All isolates toxic in HeLa cells were also toxic in McCoy cells . The prevalence of cytotoxic strains was not significantly different between the endoscopic findings; 50% in normal endoscopy, 60% in non-ulcer dyspepsia and 59% in peptic ulcer disease . However, cytotoxic strains were more common in subjects with severe histological gastritis than in those with normal mucosa or mild gastritis (66% vs 30%, p<0.01) . Similarly, the prevalence of cytotoxic strains was also higher in subjects with active gastritis than in those without (64% vs 23%, p<0.01) . CONCLUSIONS: McCoy cells are more sensitive than HeLa cells for detecting Helicobacter pylori cytotoxicity in vitro . There is an association between cytotoxic strains and the severity and activity of histological gastritis.

Acta Otolaryngol, 1999, 119(8), 939 - 43
Immunoglobulin-secreting cells in the surface secretion on the pharyngeal tonsils; Ivarsson M et al.; As B-lymphocytes on the pharyngeal tonsils constitute a considerable part of the leukocytes in the surface secretion, and their biological role is obscure, we explored their possible function with respect to immunoglobulin production . Twenty children scheduled for routine adenoidectomy participated . Surface secretion from 10 children was analysed for presence of plasma cells and cells from the secretions of the other 10 children were tested in enzyme-linked immunosorbent spot assays (ELISPOT-assays) for their capacity to secrete and produce IgA, IgM and IgG . Plasma cells and cells that secreted IgA, IgM and IgG respectively were present in the secretions of all tested children . In eight of ten children the IgG immunocytes, Ig-producing blasts and plasma cells . outnumbered the IgA immunocytes . The number of immunoglobulin secreting cells (ISCs) was reduced by half or more in cell suspensions exposed to the reversible protein synthesis inhibitor cycloheximide . It is concluded that immunocytes that produce and secrete immunoglobulin are present in the surface secretion on the pharyngeal tonsils . The production represents an addition to the immunoglobulins transported to the secretion by the poly-Ig receptor and by passive diffusion . The results shed new light on the pathogenesis of mucosal infections in the upper airways.

Methods Cell Sci, 1999, 21(2-3), 141 - 8
Establishment of synchronization in carrot cell suspension culture and studies on stage specific activation of glyoxalase I; Ghosh S et al.; The present report summarizes and compares the effects of three cell cycle inhibitors, viz . aphidicolin, hydroxyurea and mimosine, in inducing synchronization of a rapidly proliferating suspension culture of carrot . These treatments efficiently synchronized the cell cycle as the doubling time of the cell population was roughly equal to the total length of one cell cycle . Protoplasts derived from mimosine treated cell suspension culture were resolved via flow cytometry to get an idea of the temporal organization of the cell cycle events . The biochemical analysis showed a rise in stage specific activity of glyoxalase I, an auxin inducible marker enzyme activated at G2-M . This activity peak could be shifted to an early phase of interphase in response to auxin treatment.

Methods Cell Sci, 1999, 21(2-3), 109 - 21
Study of phase-specific gene expression in synchronized tobacco cells; Combettes B et al.; Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division . In the last few years, considerable progress has been achieved in identifying the major components of the cell cycle in plants . The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and the next years . This review summarizes our current knowledge at molecular and biochemical levels of cell cycle-regulated expression in the model system, the synchronized tobacco BY2 cell suspension, and discusses the results in comparison to those obtained by different methods and in other plant systems.

J Exp Med, 2000 Mar 20, 191(6), 961 - 76
Multiple genetic alterations cause frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer; Koopman LA et al.; The nature and frequency of human histocompatibility leukocyte antigen (HLA) class I loss mechanisms in primary cancers are largely unknown . We used flow cytometry and molecular analyses to concurrently assess allele-specific HLA phenotypes and genotypes in subpopulations from 30 freshly isolated cervical tumor cell suspensions.Tumor-associated HLA class I alterations were present in 90% of the lesions tested, comprising four altered pheno/genotype categories: (a) HLA-A or -B allelic loss (17%), mostly associated with gene mutations; (b) HLA haplotype loss, associated with loss of heterozygosity at 6p (50%) . This category included cases with additional loss of a (third) HLA-A or -B allele due to mutation, as well as one case with an HLA class I-negative tumor cell subpopulation, caused by a beta2-microglobulin gene mutation; (c) Total HLA class I antigen loss and retention of heterozygosity (ROH) at 6p (10%); and (d) B locus or HLA-A/B downregulation associated with ROH and/or allelic imbalance at 6p (10%) . Normal HLA phenotypes and ROH at 6p were observed in 10% of the cases . One case could not be classified (3%).Altered HLA class I antigen expression occurs in most cervical cancers, is diverse, and is mainly caused by genetic changes . Combined with widespread tumor heterogeneity, these changes have profound implications for natural immunity and T cell-based immunotherapy in cervical cancer.

Phytochemistry, 2000 Mar, 53(5), 533 - 8
Purification and characterization of UDP-glucuronate: baicalein 7-O-glucuronosyltransferase from Scutellaria baicalensis Georgi . cell suspension cultures; Nagashima S et al.; UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to the 7-OH of baicalein . UBGAT was purified from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae) . It was purified 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity . The Mr was estimated to be 110 kDa by gel filtration chromatography with a 52 kDa subunit by SDS-PAGE . The isoelectric point was pH 4.8 . UBGAT was specific to UDP-glucuronic acid as a sugar donor and flavones with substitution ortho- to the 7-OH group such its baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe).

J Biol Chem, 2000 Mar 24, 275(12), 8404 - 8
A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium; Schaloske R et al.; Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release . In this study we show that this is also true for Dictyostelium . In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release . This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J . A . , Lam, T . H., Catlin, M . C., Costa, L . G., Molinski, T . F., and Pessah, I . N . (1997) Neuron 19, 723-733) . In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal {Ca(2+)}(i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions . Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.

Brain Res Mol Brain Res, 2000 Mar 10, 76(1), 121 - 31
GFAP mRNA positive glia acutely isolated from rat hippocampus predominantly show complex current patterns; Zhou M et al.; Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats . As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention . However, the actual classification of these electrophysiologically complex glial cells remains unclear . They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100beta, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP) . To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats . In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells) . We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA . We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive . We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively . In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP . The expression of GFAP at the transcript level indicates GFAP (-)/GFAP mRNA (+) glial cells have an astrocytic identity . As single cell RT-PCR is able to detect both GFAP (-)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.

Brain Res Bull, 2000 Feb, 51(3), 203 - 11
Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects; Kaddis FG et al.; Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro . To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum . In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM) . Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling . These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.

Brain Res Bull, 2000 Mar 15, 51(5), 371 - 8
Enhancing effect of electric stimulation on cytotoxicity of anticancer agents against rat and human glioma cells; Horikoshi T et al.; Electropermeabilization, or electroporation, has been used to deliver genes or drugs into the cytoplasm through micropores in the cell membrane caused by electric stimulation . The cytotoxic effect of a combination of anticancer agents with electric stimulation on rat C6 and human T98G glioma cells was examined in vitro . Electric pulses of 100 microsec square waves (eight cycles at 1 Hz) at various electric fields were delivered to C6 or T98G glioma cell suspensions in combination with several anticancer agents . Cell growth was evaluated 48-72 h after treatment . Measurement of cell lysis by electric stimulation was used to assess the optimum field strength for electroporation . Electric stimulation enhanced significantly the cytotoxicity of bleomycin to both C6 and T98G cells by more than 1000-fold using an electric field of 1750 V/cm for C6 cells and 1000 V/cm for T98G cells . The enhancement disappeared when bleomycin concentration was reduced to 100 pg/ml . The cytotoxicity of carboplatin was weakly but significantly enhanced by electric stimulation when a high dose of carboplatin was used . However, there was no enhancement of the cytotoxicity of nimustine hydrochloride (ACNU), etoposide, and vincristine . These results indicate that the combination of bleomycin and electroporation is the most potent candidate for electrochemotherapy in vivo.

J Virol Methods, 2000 Apr, 86(1), 85 - 94
An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method; Schirawski J et al.; An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented . This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time . These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection . The simple transfection procedure has been optimized at various steps . Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

Plant Physiol, 2000 Mar, 122(3), 793 - 801
Detoxification of arsenic by phytochelatins in plants; Schmoger ME et al.; As is a ubiquitous element present in the atmosphere as well as in the aquatic and terrestrial environments . Arsenite and arsenate are the major forms of As intoxication, and these anions are readily taken up by plants . Both anions efficiently induce the biosynthesis of phytochelatins (PCs) ({gamma-glutamate-cysteine}(n)-glycine) in vivo and in vitro . The rapid induction of the metal-binding PCs has been observed in cell suspension cultures of Rauvolfia serpentina, in seedlings of Arabidopsis, and in enzyme preparations of Silene vulgaris upon challenge to arsenicals . The rate of PC formation in enzyme preparations was lower compared with Cd-induced biosynthesis, but was accompanied by a prolonged induction phase that resulted finally in higher peptide levels . An approximately 3:1 ratio of the sulfhydryl groups from PCs to As is compatible with reported As-glutathione complexes . The identity of the As-induced PCs and of reconstituted metal-peptide complexes has unequivocally been demonstrated by electrospray ionization mass spectroscopy . Gel filtration experiments and inhibitor studies also indicate a complexation and detoxification of As by the induced PCs.

Immunol Invest, 2000 Feb, 29(1), 61 - 70
Kinetics of peptide uptake and tissue distribution following a single intranasal dose of peptide; Metzler B et al.; We have previously used intranasal (i.n.) peptide application to induce mucosal tolerance in experimental autoimmune encephalomyelitis (EAE) . This strategy, however, appeared to give rise to similar phenomena of tolerance observed as a result of systemic administration of soluble antigenic peptide . We were interested, therefore, in the uptake and tissue distribution of peptide following i.n . treatment . In the H-2u mouse model of EAE, the highly tolerogenic peptide analogue Ac1-9{4Y} of myelin basic protein (MBP) displays high affinity binding to Au MHC class II . For the purpose of the present study this peptide was synthesised to contain a tritiated acetyl group and a protocol was developed to recover radioactivity in solubilised tissues taken at various times after {3H}Ac1-9{4Y} i.n. . Radiolabel loads of the lung and gastro-intestinal tract were initially high but declined rapidly . Radiolabel uptake by blood and lymphoid tissues followed similar kinetics with peak levels around 2.5-4 hours after i.n . administration . Concentrations were high in the draining cervical lymph nodes (CLN) but also reached significant levels in the spleen and 'nondraining' inguinal lymph nodes . The presence of intact antigenic peptide was demonstrated in spleens and CLN from Ac1-9{4Y} i.n . treated mice . Cell suspensions prepared from these tissues at selected time points after peptide i.n . were able to stimulate peptide-specific T cell lines up to at least one day after peptide i.n., suggesting long lasting formation of stable Au-Ac1-9{4Y} complexes in vivo.

J Steroid Biochem Mol Biol, 1999 Dec 31, 71(5-6), 203 - 11
Uptake of dehydroepiandrosterone-3-sulfate by isolated trophoblasts from human term placenta, JEG-3, BeWo, Jar, BHK cells, and BHK cells transfected with human sterylsulfatase-cDNA; Ugele B et al.; The human placenta lacks the enzyme 17alpha-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens . Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood . To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of {(3)H}-/{(35)S}-DHEA-S and {(3)H}-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied . Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured . During the first 5 min of incubation with {(3)H}-DHEA-S or {(35)S}-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase . Radioactivity of BeWo cells increased slightly . For all cell types, there was no significant difference for uptake of either substrate . During incubation with {(3)H}-taurocholate, radioactivity of cytotrophoblasts did not increase . Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension . Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension . In BHK and Jar cells sterylsulfatase activity was not detectable.Cytotrophoblasts take up DHEA-S without prior hydrolysis . BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S . In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited . Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides.

J Immunol Methods, 2000 Mar 6, 236(1-2), 27 - 35
Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers; Van Damme N et al.; Chemical agents (DTT, EDTA) and enzymes (collagenase, DNAse) are often used for the generation of a single cell suspension from tissue samples . In this study, flow cytometry was used to examine the effect of chemical agents and enzymes on the expression of cell membrane markers of T lymphocytes from tonsils and peripheral blood . Expression of CD4, CD8, CD25, CD38, L-selectin, CD44, alphaEbeta7 and alpha4beta7 were studied . Incubation of lymphocytes with DTT and EDTA resulted in a decrease of CD38, alphaEbeta7 and alpha4beta7 expression . Incubation with collagenase A and DNAse resulted in a decrease of CD25, L-selectin, alphaEbeta7 and alpha4beta7 . The results of this study indicate that a careful interpretation is necessary for phenotypic descriptions of lymphocyte populations obtained by enzymatic isolation techniques.

Strahlenther Onkol, 2000 Feb, 176(2), 81 - 3
Comparison of radiation effects of gadolinium and boron neutron capture reactions; Tokuuye K et al.; PURPOSE: Cell survival assays were performed to evaluate the effects of radiations released during neutron capture reactions by gadolinium-157, boron-10 and by the combination of both . MATERIALS AND METHODS: Single cell suspensions with or without Gd-157 and/or B-10 were exposed to thermal neutrons produced by the Kyoto University reactor, and standard cell survival curves were obtained . RESULTS: Under the same molarity, cytocidal effects were 1.5 times greater for Gd-157 than for boron when compared at 10% survival levels . The presence of B-10 enhanced the radiation effect of Gd-157 neutron capture by 1.2-fold, suggesting that cells were not sufficiently irradiated as a result of neutron fluency attenuation by the presence of excess neutron capture agents in the medium . CONCLUSIONS: When an equal number of atoms were present, Gd-157 was effective as B-10 when exposed to an equal number of thermal neutrons . However, there was no benefit observed in the combination of Gd-157 and B-10 for neutron capture therapy . Further studies are needed to determine optimal Gd-157 and B-10 concentrations as a function of tumor dimension.

Free Radic Res, 1999 Dec, 31 Suppl, S19 - 24
Ozone detoxification in the mesophyll cell wall during a simulated oxidative burst; Moldau H; With the aim to assess the effect of possible O2*- generation during an oxidative burst on O3 reduction in mesophyll cell walls due to the reaction O2*- + O3 --> O3*- + O2 and subsequent formation of *OH, 03 flow through this sequence was compared with O2*- flow through the competitive sequences where H2O2 is formed . The two-electron reduction of O3 via the direct reaction with ascorbate was also considered . The calculations were exemplified in an experiment where Phaseolus vulgaris L . leaves were exposed to 530 nl O3 l(-1) in air for 3.5 h . During the exposure, H2O2 was assumed to be generated at peak rates observed in pathogen-elicited cell suspensions . O3 reduction through reaction with O2*- was 25-44% of O3 detoxified in the direct reaction with ascorbate . More than 99% of O2*- was reduced to H2O2 via spontaneous disproportionation and reduction with ascorbate, the disproportionation prevailing at pH 5 and reduction at the expense of ascorbate at pH 7 . H2O2 was estimated to be channelled mostly to the peroxidase-catalysed scavenging reaction . Calculated steady state H2O2 concentrations were 40-80 microM . It is concluded that generation of H2O2 at the postulated rate was too high and that the generation of O2*- during an oxidative burst is ineffective in reducing O3 through the network of reactive oxygen species . Superoxide dismutase induction in the cell wall under O3 is discussed.

Eur J Biochem, 2000 Mar, 267(5), 1397 - 406
The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha/betahydrolase super family; Dogru E et al.; The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes . In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton . PNAE was purified from cell suspension cultures of Rauvolfia serpentina . The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R . serpentina library . The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography . The pure enzyme shows extraordinary substrate specificity, completely different to other esterases . Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.

J Nat Prod, 2000 Feb, 63(2), 185 - 9
Biosynthesis of 4'-O-methylpyridoxine (Ginkgotoxin) from primary precursors; Fiehe K et al.; Cell suspension cultures of Ginkgo biloba and Albizia tanganyicensis were investigated for the presence of 4'-O-methylpyridoxine (ginkgotoxin, 2), the 4'-O-methyl derivative of vitamin B(6) (pyridoxine, 1) . The cultures produced the toxin even in the absence of vitamin B(6) (a common additive to plant cell culture media) . This indicates that the pyridoxine ring system of ginkgotoxin is synthesized de novo by the cultured cells . A feeding experiment with D-{U-(13)C(6)}glucose revealed that the mode of incorporation of label into the pyridoxine moiety of 2 matched that observed for 1 in Escherichia coli . Thus, the data obtained in this investigation provide independent proof supporting the current hypothesis on vitamin B(6) biosynthesis . The 4'-O-methyl group of ginkgotoxin (2) was labeled from L-{methyl-(13)C(1)}methionine . This indicates that ginkgotoxin is likely to be derived by 4'-O-methylation of pyridoxine (1) . The G . biloba cell suspension culture may be a suitable system to get further insight into vitamin B(6) and/or ginkgotoxin biosynthesis.

Int J Oral Maxillofac Surg, 2000 Feb, 29(1), 36 - 41
Nuclear DNA content, an adjunct to p53 and Ki-67 as a marker of resistance to radiation therapy in oral cavity and pharyngeal squamous cell carcinoma; Raybaud H et al.; Factors of prognosis and radioresistance in oral cavity and pharyngeal squamous cell carcinoma (OCPSCC) are limited . In the present study, the usefulness of tumor DNA content in predicting radioresistance in patients with OCPSCC has been investigated . Radioresistance has been defined as local recurrence or tumor persistence after radiation therapy . DNA-ploidy analysis was performed by static cytometry on smears of cell suspensions obtained from formalin-fixed paraffin-embedded material and stained with Feulgen . DNA-ploidy was correlated with the proliferation rate (Ki-67) and p53 protein accumulation obtained by immunohistochemistry . The follow-up of patients ranged from 8 to 62 months . Radioresistance was more common in non-diploid tumors; 14/28 (50%) non-diploid tumors recurred, whereas only 3 (10.7%) out of 28 diploid tumors had local failure (P=0.0019) . Proliferation rate and p53 accumulation, evaluated by immunohistochemistry, also added prognostic information . Twelve out of 14 failures were from non-diploid tumors with a low proliferation rate (Ki-67<20%), whereas none of 20 p53-negative diploid tumors developed recurrences . This study showed that non-diploid tumors responded poorly to radiotherapy . DNA content appeared, therefore, as a significant prognostic marker for the evaluation of OCPSCC in patients receiving radiation therapy . This study also showed that DNA content adds information to p53 accumulation and the proliferation rate (Ki-67) for the purposes of determining patient management.

Vet Immunol Immunopathol, 2000 Feb 25, 73(2), 145 - 54
Identification of Ostertagia ostertagi specific cells in bovine abomasal lymph nodes; De Marez T et al.; To investigate the contribution of different bovine cell subpopulations in the development of in vitro induced responses by Ostertagia ostertagi third larval antigen extract (L3), bovine abomasal lymph node cell suspensions were depleted of specific cell populations . The depleted cell suspensions were subsequently assayed for their proliferative responses to O . ostertagi L3 antigen extract . Proliferative responses to O . ostertagi L3 antigen extract were restricted to a CD2+ CD4- CD8- cell population and MHC II+ cells different from B-cells were of major importance . Depletion of CD4, CD8, CD4CD8, IgM or CD21 positive cells did not decrease proliferation to L3 antigen extract . Depletion of gammadelta T-cells, which also comprise a subpopulation of CD2+ CD4- CD8- cells, reduced proliferation to L3 antigen extract only in one animal . The results suggest that either gammadelta T-cells could be involved in the proliferation or that another as yet unidentified population is important for proliferation . The precise role of these populations during infection with O . ostertagi and the mechanism by which these cells may influence the host immune response are important issues that remain to be elucidated.

Eur J Pharmacol, 2000 Feb 18, 389(2-3), 131 - 40
The effect of protein kinase C activation on colonic epithelial cellular integrity; Tepperman BL et al.; We have investigated whether activation of protein kinase C has a direct cytotoxic effect on colonic mucosal epithelial cells and whether oxidant-induced damage to colonocytes is mediated by activation of cellular protein kinase C . Incubation of freshly harvested cells from rat colon with the protein kinase C activator, phorbol 12-myristate, resulted in a concentration-dependent increase in the extent of cell injury . Phorbol 12-myristate acetate (0.1-10 microM) also increased cellular protein kinase C activity and this was reduced significantly by treating cells with the antagonists staurosporine or 2-{1-(3-dimethylaminopropyl)-indol-3-yl}3-(-indol-3-yl)maleimide (GF 109203X; 10 microM) . Phorbol 12-myristate acetate treatment also resulted in increased translocation of proteins for protein kinase C isoforms alpha, delta and epsilon from cytosol to membrane particulate fractions . The antagonists reduced the extent of cell damage in response to phorbol 12-myristate acetate . Furthermore, cell injury in response to the phorbol acetate was also inhibited by the addition of the oxidant scavengers, superoxide dismutase or catalase to the cell suspension . Addition of H(2)O(2) to the incubation medium (0.1-100 microM) resulted in an increase in cellular protein kinase C activity, an increase in the expression of the alpha, beta and zeta isoforms and a reduction in cell integrity . The cellular damaging actions of H(2)O(2) were significantly reduced by the protein kinase C antagonists, staurosporine or 2-{1-(3-dimethylaminopropyl)-indol-3-yl}-3-(-indol-3-yl)maleimide (GF 109203X) . These findings suggest that protein kinase C activation results in colonic cellular injury and this damage is mediated, at least in part, by release of reactive oxidants . Furthermore, oxidant-mediated damage to these cells also involves protein kinase C activation.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 259 - 264
Growth characteristics and chemical analysis of Psychotria carthagenensis cell suspension cultures; Lopes SO et al.; Callus and cell suspension cultures of Psychotria carthagenensis have been established in Gamborg's B5 medium supplemented, respectively, with 3% sucrose, 0.2 mg/l kinetin, and 1.0 mg/l 2,4-D and 2% sucrose, 2.0 mg/l 2,4-D, 0.2 mg/l kinetin, and 50 mg/l cysteine . Suspension culture presented a typical growth curve with the complete cycle of ca . 18 days and the maximum specific growth rate (micro) was 0.0099 day . The presence of different secondary metabolite pathways was determined by measuring the enzyme activity of phenylalanine ammonia lyase (PAL), tryptophan decarboxylase (TDC), strictosidine synthase (STR), strictosidine-beta-glucosidase (SG), and geraniol-10-hydroxylase (G10H) . Activity could only be measured for SG (14.55 pkatal/mg protein) and G10H (0.3 pkatal/mg protein) . Analysis of extracts from leaves, callus and cell suspension cultures demonstrated the presence of two major triterpenes: beta-sitosterol and ursolic acid.(2)

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 229 - 234
Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin; Gilleta F et al.; Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads . Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin . On the contrary, immobilized plant cells excreted considerable amounts of scopolin . Scopolin diffused throughout the gel matrix and reached the culture media . A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells . Immobilized N . tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass {into the culture media} versus 0.2 mg/g fresh weight biomass {intracellular}) . Variation of the immobilization conditions revealed a marked influence on the behavior of N . tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies . This excretion phenomenon could be used advantageously at an industrial production level.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 222 - 228
Recombinant Escherichia coli cell for d-p-hydroxyphenylglycine production from d-N-carbamoyl-p-hydroxyphenylglycine; Fan C et al.; Recombinant Escherichia coli cell containing D-amidohydrolase was employed to convert D-N-carbamoyl-p-hydroxyphenylglycine (D-CpHPG) to D-p-hydroxyphenylglycine (D-pHPG) . Biotransformations under pH 7 and 40 degrees C allowed to complete conversion of D-CpHPG into D-pHPG . Under the same reaction pH, the D-amidohydrolase activity of the cell in the phosphate buffer was higher than that in the Tris buffer . The activity decreased with the increase of phosphate buffer concentration . Instead of using buffer, the reaction pH maintained constant at 7 by titrating with 1 N HCl resulted in a higher D-pHPG production rate . Flocculating the cell suspension with chitosan and cross-linked by glutaraldehyde made the cell recovery for repeated use much easier . Both the cross-linking and (PMSF; a protease inhibitor) treatments could increase the cell reusability and storage stability . However, the cross-linking decreased the D-amidohydrolase activity of the cell to about 50% . The D-amidohydrolase activities of free and cross-linked cell were inhibited at substrate concentration higher than 150 mM and 100 mM, respectively . The conversion of 150 mM D-CpHPG to D-pHPG could be completed within 7 h for the free cell at the concentration of 10% (wet weight/volume).

Hua Xi Yi Ke Da Xue Xue Bao, 1997 Mar, 28(1), 37 - 9
{Alkaloid production of cultured coptis cells by two-stage suspension-culture}; Zhang H et al.; In this study the asexual cell line H292 induced and selected from Coptis gulinensis had rapid growth rate and could stably produce alkaloids . By the one-stage method, after the cell suspensions were cultured on the same medium for six weeks, the increased dry and fresh weights of the cells were 20.96 g/L and 174.92 g/L respectively . The content of the total alkaloids in the cells was 14.79 mg/g cell dw . Per litter liquid medium could provide 323 mg alkaloid . In contrast, the cells were cultured by two-stage method . After having been cultured on the medium which contributed to the growth of the cells for three weeks, the cells were transferred to the medium which contributed to the production of the alkaloid and cultured for three weeks . Six weeks later, the dry and fresh weights of the cells were 16.72 g/L and 127.44 g/L, respectively . The biomass was lower than that by one-stage method, but the content of the total alkaloids was as high as 31.76 mg/g cell dw, which was much more than that by one-stage method . In addition, the content of the alkaloid in the medium was 25.31 mg/L . Per litter liquid medium could provide 556 mg alkaloid . The total yield of alkaloid obtained by two-stage method was 1.72 times than that by one-stage method.

J Cardiovasc Pharmacol Ther, 1999 Jan, 4(1), 41 - 48
Mechanisms of Hydrogen Peroxide-Induced Increase in Intracellular Calcium in Cardiomyocytes; Wang X et al.; BACKGROUND: Hydrogen peroxide (H(2)O(2)) in high concentrations has been implicated in heart dysfunction attributable to ischemia-reperfusion . Although H(2)O(2) is also known to increase the intracellular concentration of Ca(2+) ({Ca(2+)}(i)) in cardiomyocytes, the mechanisms for such a change are not clear . In this study, the sources and mechanisms of increase in {Ca(2+)}(i) caused by high concentrations of H(2)O(2) in cardiomyocytes were explored . METHODS AND RESULTS: Cardiomyocytes were isolated from adult male Sprague-Dawley rats . Cell viability was examined by trypan blue exclusion test . {Ca(2+)}(i) was measured by employing cell suspension at room temperature and Fura-2 fluorescence technique . Incubation of cells with 0.25-l mmol/L H(2)O(2) increased {Ca(2+)}(i) in a time- and concentration-dependent manner . Catalase attenuated the H(2)O(2)-induced increase in {Ca(2+)}(i) significantly, whereas mannitol showed no effect . Neither the presence of verapamil, a sarcolemmal Ca(2+) channel blocker, nor the removal of Ca(2+) from the medium produced any significant reduction in the H(2)O(2)-induced increase in {Ca(2+)}(i) . Conversely, treatment of cardiomyoctes with staurosporin, a protein kinase C inhibitor, thapsigargin, a sarcoplasmic reticulum Ca(2+)-pump adenosine triphosphatase inhibitor, as well as ryanodine, a sarcoplasmic reticulum Ca(2+)-release channel blocker, markedly prevented the 0.5-mmol/L H(2)O(2)-induced increase in {Ca(2+)}(i) . The responses of cardiomyoctes to H(2)O(2) and other Ca(2+)-mobilizing agents, such as KCl or adenosine triphosphate, were additive . No changes in cardiomyocyte viability were seen on incubation with 0.5 and 1 mmol/L H(2)O(2) . Perfusion of the isolated heart with H(2)O(2) (0.1-0.5 mmol/L) depressed the left ventricular developed pressure, rate of contraction, and rate of relaxation, whereas the left ventricular end-diastolic pressure was increased . CONCLUSIONS: These results indicate that formation of H(2)O(2) under pathophysiological conditions such as ischemic heart disease may induce changes in Ca(2+) homeostasis in cardiomyocytes and may induce contractile dysfunction . Furthermore, the sarcoplasmic reticulum involving a protein kinase C-mediated mechanism appears to be the main site of action of H(2)O(2) in cardiomyocytes.

J Cardiovasc Pharmacol Ther, 1998 Oct, 3(4), 291 - 298
Modification of the ATP-Induced Increase in
Musat S, Wang X, Dhalla NS.
BACKGROUND: It is generally accepted that the plasma membrane of mammalian ventricular myocytes regulates the cytosolic concentration of Ca(2+) . In this study we investigated the effects of some P2-purinoceptor antagonists and metals such as copper and zinc on the adenosine triphosphate (ATP)-induced increase in intracellular concentration of free Ca(2+) ({Ca(2+)}(i)) . METHODS AND RESULTS: Cardiomyocytes were isolated from adult male Sprague-Dawley rats loaded with Fura-2, and fluorescence measurements were performed by employing stirred cell suspensions at room temperature . ATP (50 microM) increased {Ca(2+)}(i) over the basal value, and 10 microM cibacron blue or verapamil virtually abolished it . The ATP-induced increase in {Ca(2+)}(i) was not observed in Ca(2+)- or Mg(2+)-free buffers . Incubation of cells with ZnCl(2) produced a significant depression of the ATP-induced increase in {Ca(2+)}(i); 25 microM Zn(2+) decreased the peak response to approximately 50% of the control value . The ATP-induced increase in {Ca(2+)}(i), was inhibited by low concentrations (1-5 microM) of Cu(2+) but was markedly augmented by high concentrations (25 microM) of Cu(2+) . The increase in the {Ca(2+)}(i) response to cron blue, and Zn(2+), but not by ryanodine or caffeine pretreatment . CONCLUSIONS: The ATP-induced increase in {Ca(2+)}(i) is dependent on the extracellular concentrations of Ca(2+) as well as Mg(2+) and is antagonized by cibacron blue and Zn(2+) . On the other hand, Cu(2+) produced a biphasic response to the ATP-induced increase in {Ca(2+)}(i) in cardiomyocytes.

FEBS Lett, 2000 Jan 28, 466(2-3), 213 - 8
Induction of tcI 7, a gene encoding a beta-subunit of proteasome, in tobacco plants treated with elicitins, salicylic acid or hydrogen peroxide; Etienne P et al.; We previously isolated, by differential display and 5' RACE (rapid amplification of cDNA ends), cDNAs corresponding to genes activated following cryptogein treatment of tobacco cell suspensions, among them tcI 7 (tcI for tobacco cryptogein Induced), a gene encoding a beta-subunit of proteasome . Here, we report that tcl 7 was up-regulated in tobacco plants treated with elicitins (cryptogein and parasiticein) that have been shown to induce a systemic acquired resistance (SAR) . Moreover, subsequent inoculation of tobacco with the pathogen Phytophthora parasitica var . nicotianae (Ppn) was shown to induce an additional activation of tcI 7 in tobacco plants pretreated with cryptogein . We also showed an up-regulation of tcI 7 by salicylic acid (SA) . Moreover, accumulation of tcI 7 transcripts after treatment with cryptogein or with SA only occurred in NahG 9-tobacco plants that do not express the salicylate hydroxylase and thus are able to accumulate SA and develop a SAR . Suppressed accumulation of tcI 7 transcripts in NahG 8+ tobacco plants after cryptogein or SA treatment correlated with the loss of SAR . H2O2 was also shown to up-regulate tcI 7 in tobacco plants . Using gene walking by PCR we cloned and sequenced the 5' flanking region of tcI 7 containing hypothetical regulatory sequences, especially myb and NF-kappaB boxes, that could be responsible for the regulation of tcI 7 by salicylic acid and H2O2 respectively.

Phytochemistry, 2000 Jan, 53(2), 187 - 93
Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences; Arend J et al.; Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone . A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase . SDS-PAGE showed a M(r) for the enzyme of 52 kDa . Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced . Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.

Cytometry, 2000 Feb 15, 42(1), 43 - 9
Novel functional multiparameter flow cytometric assay to characterize proliferation in skin; Mommers JM et al.; Keratins are a group of cytoskeletal proteins that are found in human epidermis and other stratified squamous epithelia . Several different types of keratins have been described . Keratin 10 (K10) is a keratin that is expressed in well differentiated, suprabasal keratinocytes, and keratin 6 (K6) is a keratin which is associated with hyperproliferation . Psoriasis is a chronic inflammatory skin disease, and besides inflammation, disturbed differentiation and hyperproliferation are its hallmarks . In order to study the hyperproliferation associated keratinization in both well differentiated and poorly differentiated keratinocytes, and in order to assess the proliferative activity of all K10 and K6 subpopulations, simultaneous assessment of K6, K10, and DNA content is required . So far, a triple staining protocol had not been available . In the present study, we established a novel protocol for simultaneous measurement of K6, K10, and DNA content, which enables the characterization of the proliferative activity of several cellular subpopulations in epidermis . From 16 patients with psoriasis and from 15 healthy volunteers, punch biopsies were obtained . After preparation of single cell suspensions, cells were stained with the anti-keratin 10 IgG(1)-isotype monoclonal antibody RKSE60, with the anti-keratin 6 IgG(2a)-isotype monoclonal antibody LHK6B, and with the DNA fluorochrome TO-PRO-3 iodide . Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein isothiocyanate (FITC) were used as the second step in the staining procedure . Controls were measured omitting the primary antibodies, and gates were set in order to differentiate between the K10 and K6 subpopulations . Samples from both psoriatic patients and healthy volunteers were than measured . Owing to the IgG specificity of RKSE60 and LHK6B, no cross-reactivity was observed between these antibodies . The triple staining with RKSE60, LHK6B, and TO-PRO-3 iodide showed subpopulations of K10 expressing cells, K10/K6 co-expressing cells, and K6 only expressing cells . There was a significant difference in the proportion of K6 expression and K10/K6 co-expression between psoriatic and normal skin . Moreover, the proliferative activity of these subpopulations could be quantified by this protocol . We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using the monoclonal antibodies LHK6B, RKSE60, and TO-PRO-3 iodide, supplies reliable and reproducible data for cellular studies on these keratins and for studying the proliferative activity of the subpopulations of these keratins in epidermis . Moreover, the present study showed that with respect to the proportion of K6, significant differences are present between psoriatic and healthy human skin .

Cytometry, 2000 Feb 1, 39(2), 96 - 107
Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer; Corver WE et al.; BACKGROUND: Multiparameter DNA flow cytometry using a one-laser bench-top flow cytometer has been restricted to three different colors . The two laser FACSCalibur has recently been introduced, allowing four-color analysis . Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al . 1996) to a four-color analysis of phenotypic intra-tumor heterogeneity using a bench-top flow cytometer . METHODS: First, the effect of a range of different propidium iodide (PI) and TO-PRO-3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde-fixed lysolecithin-permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells . Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti-keratin antibodies . Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3-W vs . FL3-A) . The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity . Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell-adhesion molecule (Ep-CAM; APC fluorescence), (b) keratin (R-phycoerythrin {RPE} fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein-isothiocyanate {FITC} fluorescence) to label stromal cells . RESULTS: Overall, PI produced better CVs than did TP3 . The optimal concentration of PI was 50-100 microM for all cells tested . Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa) . Optimal TP3 concentrations were 0.25-2.0 microM . Average CVs were 2 . 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa) . Inter- or intra-DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions . Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction . CONCLUSIONS: PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells . Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules . The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories .

Cytometry, 2000 Feb 1, 39(2), 91 - 5
Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa; Lamatsch DK et al.; BACKGROUND: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a simple, noninvasive technique for DNA content and ploidy determination . METHODS: From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0 . 6-gauge needle and incubating it for 20 min at room temperature (RT) with gentle agitation . After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 min at RT before adding DAPI to a final volume of 2 ml . The cells were stained for 1-3 h and then analyzed by flow cytometry . RESULTS: We obtained good measurements with CVs ranging from 1.23% to 3.36% . The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzias latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio (zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg . All values except zebrafish are in good agreement with the literature . CONCLUSIONS: The identification of living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is now made possible . With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers .

J Biomed Mater Res, 2000 May, 50(2), 153 - 9
Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity; Shibutani T et al.; This study was designed to evaluate the use of apatite-collagen complexes (ACC) coated onto glass slides for measurement of osteoclastic resorption activity . ACC-coated glass slides were prepared by immersion in beta-glycerophosphate solution for 7-14 days after glass slides coated with type I collagen had been treated with alkaline phosphatase and phosvitin . Osteoclast-containing cell suspensions were prepared from the long bones of 1-day-old rabbits and were seeded in medium 199 (containing 10% FBS) onto ACC-coated glass slides . After allowing the cells to attach for 1.5 h, the glass slides were incubated for periods of up to 96 h . The cells were observed by scanning electron microscopy and cytochemically for tartarate resistant acid phosphatase (TRAP) activity . Some slides were treated with FITC-phalloidin and anti-type I collagen antibody . TRAP-positive multinucleated cells were located in transparent spaces on the glass slides . These spaces did not stain immunohistochemically with anti-type I collagen antibody . Podosome formation was observed in the multinucleated cells facing the edge of the transparent spaces . The scanning electron microscopy demonstrated well-spread large cells located on the flattened surface on apatite particles covering the glass surface . Our results suggest that osteoclasts could resorb the apatite particles and coated collagen on the glass slide . The resorption lacunae appeared as transparent spaces, and the cytoskeleton of resorbing osteoclasts was observed in these spaces . ACC-coated glass slides could be useful for investigating the function and metabolic activities of osteoclasts .

Ann N Y Acad Sci, 1999, 897, 273 - 81
Stimulation of alpha-amylase release in the scallop Pecten maximus by the myosuppressins . Structure-activity relationships; Nachman RJ et al.; The insect myosuppressin LMS (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2) elicits potent stimulation of the release of the digestive enzyme alpha-amylase from cell suspensions of the stomach-digestive gland complex of the scallop Pecten maximus . The myosuppressins are members of the FMRFamide-like peptide superfamily, which immunocytochemical data confirm is present in the scallop . Structure-activity studies indicated that the two most critical residues for bioactivity are Arg and Phe . Bioactivity of the peptide can be maintained if the basic, aromatic residue His is replaced by another basic residue (Lys) and another aromatic residue (Trp), but not the aromatic Tyr, indicating a sensitivity to the introduction of a phenolic OH group . A restricted-conformation analogue containing a cyclopropyl-Ala residue in position 8 (Cpa-MS) demonstrates an ability to antagonize the amylase secretion activity of LMS at microM concentrations . This result provides evidence that the myosuppressins adopt a tight turn in the C-terminal tetrapeptide active core region while binding to the scallop digestive gland receptor . Cpa-MS may provide a useful tool to neuroendocrinologists studying in vitro and in vivo digestive processes in mollusks and other invertebrates.

Int J Oncol, 2000 Mar, 16(3), 461 - 8
Microdissection based comparative genomic hybridization analysis (micro-CGH) of secondary acute myelogenous leukemias; Heller A et al.; Comparative genomic hybridization (CGH) is a well established technique in molecular cytogenetics . However, leukemias, and especially secondary acute myelogenous leukemias (sAML) are not very well analyzed by this technique, even though such diseases are often characterized by complex karyotypic changes, not resolvable by conventional cytogenetic banding analysis . This lack of CGH-studies might be due to the fact, that in most cases bone marrow aspirate is too limited to do DNA-extraction additionally to the cytogenetic analysis . To circumvent this problem a new CGH technique has been applied to analyze 10 AML cases with complex karyotypic changes . In each case 15 interphase nuclei of the harvested and fixed bone marrow cell-suspension have been microdissected from the coverslip surface and collected in a tube . Subsequently, DNA was amplified by DOP-PCR . With this micro-CGH technique additional cytogenetic information from 10 highly aberrant AML cases was obtained and confirmed by FISH on metaphase of the corresponding AML case.

J Struct Biol, 2000 Feb, 129(1), 17 - 29
Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy; Schaper A et al.; The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM) . Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried . Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM) . The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data . The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3) . A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities . Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious . Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing . We imaged intact chromosomes in liquid by SFM without any intermediate drying step . A granular surface was obvious but with an appreciably lower resolution . Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations .

J Photochem Photobiol B, 1999 Nov-Dec, 53(1-3), 121 - 7
Time-resolved detection of singlet oxygen luminescence in red-cell ghost suspensions: concerning a signal component that can be attributed to 1O2 luminescence from the inside of a native membrane; Oelckers S et al.; For about ten years, it has been debated whether in principle it is possible to detect 1O2 located within the cell membrane by performing experiments with cell suspensions or even in tissue . In this paper we present our investigations on photosensitized red-cell ghost suspensions (RCGSs) and our strategy for the detection of luminescence of singlet oxygen (1O2) from the inside of the cell membrane . Using a very sensitive apparatus for time-resolved 1O2 detection, a very promising sensitizer and an adequate experimental strategy, a very small amount of the detected luminescence indeed can be attributed to 1O2 from the inside of the ghost membrane.

Clin Endocrinol (Oxf), 2000 Feb, 52(2), 235 - 40
GIP-dependent adrenal Cushing's syndrome with incomplete suppression of ACTH; Croughs RJ et al.; ACTH-independent Cushing's syndrome may be due to the development of ectopic hormone receptors in adrenal tissue . Thus, in food-dependent Cushing's syndrome the adrenals aberrantly express receptors for gastric inhibitory polypeptide (GIP) . We present the case of a 60-year-old woman with food-dependent Cushing's syndrome whose cortisol levels increased after stimulation with CRH . In this patient with Cushing's syndrome the finding of low basal plasma cortisol levels in the late night and early morning as well as a paradoxical rise of plasma cortisol during a 7-h infusion with dexamethasone (carried out without any restriction in food intake), suggested that cortisol production was stimulated at times of food intake . Hourly measurements of plasma cortisol for 48 h revealed prominent meal-related peaks . A plasma cortisol response, elicited by oral glucose administration, could be prevented by octreotide . Plasma ACTH was low or undetectable . CRH administration was followed by a ACTH response from 3 to 16 ng/l and a plasma cortisol response from 230 to 680 nmol/l . Octreotide treatment for nearly five months induced a partial clinical and biochemical remission . Total bilateral adrenalectomy was performed . The left adrenal was grossly enlarged (7 x 5.5 x 4 cm) and the right adrenal was slightly enlarged (6 x 4 x 1.8 cm) . Microscopy revealed bilateral nodular hyperplasia . Cell suspensions of adrenal tissue from the patient did respond in a dose-dependent fashion to stimulation with GIP and were very sensitive to stimulation with synthetic ACTH1-24 . However, CRH had no significant effect on cortisol production in vitro . Using RT-PCR amplification and cDNA hybridization, GIP receptor was found to be overexpressed in the left and right adrenal tissues from this patient as compared to adrenal tissues from a normal individual or from non GIP-dependent adrenal Cushing's syndrome . There was no evidence of presence of adrenal CRH receptors . Thus, in this patient with food-dependent Cushing's syndrome, the CRH-induced plasma ACTH and cortisol response is probably mediated by an incomplete suppression of the HPA axis as a result of the intermittent food-dependent nature of Cushing's syndrome.

BJU Int, 2000 Feb, 85(3), 326 - 9
Apoptosis in the erectile tissues of diabetic and healthy rats; Alici B et al.; OBJECTIVE: To compare the frequency of apoptosis in the erectile tissue of chronic diabetic and healthy rats . MATERIALS AND METHODS: Fourteen chronic diabetic and 10 healthy Sprague-Dawley rats were killed, their penises harvested and stored at -70 degrees C until staining and flow cytometric analysis for apoptosis . A cell suspension was obtained from the penile tissue by scraping the inside of the cavernosum with a scalpel and filtering through a mesh . Samples of the cell suspension (0.5 x 106 cells) were stained with Annexin V (an indicator of apoptosis) and propidium iodide (PI, which stains dead cells), incubated for 15 min at room temperature and analysed by flow cytometry . The DNA content was also analysed in each sample . RESULTS: In normal erectile tissue, a mean of 6.2% of cells were stained with Annexin V, while only 2.7% were stained with PI; DNA content analyses showed 7.5% were hypodiploid cells . In diabetic rats 19.5% of cells were stained with Annexin V and 5.2% with PI; 22.9% of cells were hypodiploid . CONCLUSION: The ratio of apoptotic cells in the erectile tissues of diabetic rats was significantly greater than in normal rats . The high rate of apoptosis in diabetic rats may play a role in the pathophysiology of erectile dysfunction.

Oncol Rep, 2000 Mar-Apr, 7(2), 299 - 303
DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells; Macann AM et al.; Follicle center cell lymphoma is among the most radioresponsive of human cancers . To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs . Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay . The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay . Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line . The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells . Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma . These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs . Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.

Comp Biochem Physiol B Biochem Mol Biol, 1999 Dec, 124(4), 363 - 9
A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis; Galgani F et al.; A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601-893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum . This procedure involves the use of all tissues of P . linearis and live cell suspensions of H . ostreria or S . costatum, as DNA templates, without any further purification of DNA . As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species . Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene . The method was then used to screen mutations in partial psbA genes of 23 samples of P . linearis collected at four different stations along the mid-Atlantic coast of France . An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance . All mutations found were silent . However, genetic polymorphism discriminated between samples collected from two stations . The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae . Its application to other genes and species is considered.

Planta, 2000 Jan, 210(2), 312 - 7
Geranylhydroquinone 3"-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures; Yamamoto H et al.; Geranylhydroquinone 3"-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb . et Zucc . (Boraginaceae) . The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin . Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3", i.e . at the methyl group involved in the cyclization reaction . The enzyme is membrane-bound and was found in the microsomal fraction . It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO . The inhibitory effect of CO is reversed by illumination . These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase . The optimum pH of GHQ 3"-hydroxylase is 7.4, and the apparent K(m) value for GHQ is 1.5 microM . The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone.

Planta, 2000 Jan, 210(2), 261 - 8
Characterisation of extracellular polysaccharides from suspension cultures of members of the poaceae; Sims IM et al.; Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L . and Oryza sativa L . showed that they were comprised of numerous root primordia . Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides . In contrast, suspension-cultured cells of Hordeum vulgare L . contained mostly undifferentiated cells more typical of plant cells in suspension culture . The polysaccharides secreted by H . vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan . The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material . The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0 . From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Phytochemistry, 2000 Jan, 53(1), 7 - 12
Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450; Yamamoto H et al.; Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica . The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450 . Of the substrates examined, only specificity for loganin was demonstrated . A possible reaction mechanism is described.

J Biotechnol, 2000 Jan 21, 76(2-3), 185 - 95
Effects of Pluronic F-68 on Tetrahymena cells: protection against chemical and physical stress and prolongation of survival under toxic conditions; Hellung-Larsen P et al.; The effects of the non-ionic surfactant Pluronic F-68 (0.01% w/v) on Tetrahymena cells have been studied . A marked protection against chemical and physical stress was observed . The chemical stress effects were studied in cells suspended in buffer (starvation) or in buffers with added ingredients from a chemically defined medium (Ca2+, Mg2+, Na+, K+, trace metal ions) . The physical stress was due to mechanical stress or hyperthermia . The data show that Pluronic: (a) prolongs the survival of low concentration cell suspensions during starvation; (b) prevents the cell death caused by low concentrations of Ca2+ (70 microM); (c) prolongs the survival of cells exposed to higher ion concentrations (10 mM Ca2+, or Na+ or K+); (d) postpones the death caused by trace metal ions like Zn2+, Fe3+ and, Cu2+; (e) protects cells from the death caused by shearing forces; and (f) prolongs the survival of cells exposed to hyperthermia (43 degrees C) . The cellular survival is increased at reduced temperatures (e.g . 4 degrees C instead of 36 degrees C) and at increased cellular concentrations (e.g . 100 cells ml(-1) instead of 25 or 10 cells ml(-1)) . There is no effect of pre-incubation with Pluronic . The protective effect of Pluronic towards Tetrahymena is observed for concentrations in the range from 0.001 to 0.1% w/v.

Biorheology, 1998 Nov-Dec, 35(6), 437 - 48
A mechanism for erythrocyte-mediated elevation of apparent viscosity by leukocytes in vivo without adhesion to the endothelium; Helmke BP et al.; In spite of the relatively small number of leukocytes in the circulation, they have a significant influence on the perfusion of such organs as skeletal muscle or kidney . However, the underlying mechanisms are incompletely understood . In the current study a combined in vivo and computational approach is presented in which the interaction of individual freely flowing leukocytes with erythrocytes and its effect on apparent blood viscosity are explored . The skeletal muscle microcirculation was perfused with different cell suspensions with and without leukocytes or erythrocytes . We examined a three-dimensional numerical model of low Reynolds number flow in a capillary with a train of erythrocytes (small spheres) in off-axis positions and single larger leukocytes in axisymmetric positions . The results indicate that in order to match the slower axial velocity of leukocytes in capillaries, erythrocytes need to position themselves into an off-axis position in the capillary . In such off-axis positions at constant mean capillary velocity, erythrocyte axial velocity matches on average the axial velocity of the leukocytes, but the apparent viscosity is elevated, in agreement with the whole organ perfusion observations . Thus, leukocytes influence the whole organ resistance in skeletal muscle to a significant degree only in the presence of erythrocytes.

J Chromatogr Sci, 2000 Jan, 38(1), 33 - 7
High-performance liquid chromatographic determination of pyrazinamide in human plasma, bronchoalveolar lavage fluid, and alveolar cells; Conte JE Jr et al.; A technique is presented for the measurement of pyrazinamide in human plasma, bronchoalveolar lavage, and alveolar cells by reversed-phase column chromatography . The assay utilizes acetazolamide as an internal standard, ultraviolet detection at 268 nm, and an acetonitrile-based mobile phase . Preparation of plasma samples requires a simple deproteinization step, resulting in the development of sharp peaks with retention times of 8.4 and 17 minutes for pyrazinamide and acetazolamide, respectively . Bronchoalveolar lavage and alveolar cell suspensions require an acid extraction with ethyl acetate, evaporation to dryness, and reconstitution . This method provides specific, rapid, and reliable determinations of drug concentrations and therefore is suitable for pharmacological studies, particularly those that are designed to quantitate the intrapulmonary concentrations of pyrazinamide.

Plast Reconstr Surg, 1999 Sep, 104(4), 945 - 54
An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects; Har-Shai Y et al.; Autologous fat grafting as a technique to correct soft-tissue defects is a controversial subject . The high percentage of fat resorption and the resulting need for additional grafting considerably reduce the value of this method . The purpose of this study was to evaluate the clinical application of tissue-culturing methodology in the handling of the lipocyte aspirate in an endeavor to improve the survival rate and therefore the take of the grafted lipocytes . The method consists of syringe aspiration of the lipocytes from the donor site, isolation of intact lipocytes by gentle centrifugation, suspension of the aspirate in an enriched cell culture medium, and injection of the cell suspension into preformed subdermal tunnels . A number of media were tested and shown to prolong the survival of lipocytes ex vivo using fluorescent acridine orange stain . Implementing the integrated cell culture techniques increased the lipocytes' viability, as indicated in clinical evaluation in which the amount of graft take ranged between 50 and 90 percent . The results of 15 patients with varied types of cases who were operated on using this new methodology show that the tissue defect was filled and remained so in postoperative follow-ups of 6 to 24 months . A three-dimensional CAT scan-aided evaluation method was developed and used in one of four case histories presented herein.

Anat Histol Embryol, 1999 Dec, 28(5-6), 291 - 7
Ultrastructure of phagocytes from mammary glands of non-pregnant heifers; Sladek Z et al.; Results of ultramicroscopic investigations of phagocytes isolated from non-secreting and aberrantly secreting juvenile mammary glands of non-pregnant heifers are presented . The two types of phagocytes observed in cell suspensions obtained by lavage of mammary gland cavities were polymorphonuclear leukocytes and macrophages . Polymorphonuclear leukocytes were spherical or irregular in shape and contained segmented nuclei . Azurophilic and specific electron-dense granules, mitochondria, glycogen particles, phagosomes and phagolysosomes in cytoplasma and characteristic pseudopodia on the cell surface were observed . In addition to these normal polymorphonuclear leukocytes, degenerating cells, characterized by spherical nuclei, total absence of pseudopodia, merged nuclear segments and altered granules, other cellular organelles and plasmalemma were present . Two types of macrophages, i.e . vacuolized and non-vacuolized, could be distinguished . Typical of the non-vacuolized type was a kidney-shaped nucleus, a rich Golgi complex and a large amount of lysosomes in the cytoplasm . The vacuolized macrophages contained a large amount of electron-dense vacuoles in the cytoplasm . Unlike non-secreting glands, the cell suspensions collected from aberrantly secreting juvenile mammary glands contained only vacuolized macrophages . The vacuolization results from phagocytosis of corpuscular particles of aberrant milk plasma.

Anticancer Res, 1999 Jul-Aug, 19(4B), 3173 - 82
9-{2-(phosphonomethoxy)ethyl}-2,6-diaminopurine (PMEDAP)--a potential drug against hematological malignancies--induces apoptosis; Otova B et al.; Antitumor activity of the acyclic nucleotide analogs PMEDAP, PMEA, and PMEG was studied on a model of a spontaneous T-cell lymphoma in inbred SD/cub rats . Significant therapeutic effects were recorded after a treatment with 16 daily doses of PMEDAP at 5 mg/kg applied to the vicinity of the growing lymphoma . Identical administration of PMEA, or PMEG at a daily dose of 0.1 mg/kg did not affect the survival of lymphoma-bearing animals compared with untreated controls . A decrease in the lymphoma weight during PMEDAP administration was accompanied by the suppression of mitotic activity in neoplastic cells and increased chromatin condensation as witnessed by karyological examinations . Electron-microscopy showed the morphology of apoptotic cells (shrunken cells with condensed chromatin, apoptotic bodies) in lymphoma cell suspensions . An increase of nuclear DNA fragmentation was found during PMEDAP administration compared with spontaneous DNA fragmentation of untreated control lymphomas . These results indicate that PMEDAP application induces apoptosis in in vivo growing lymphomas . The antitumor effect of PMEDAP lasts only during the administration of the drug . After its cessation progression of neoplasia was reestablished.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 147 - 51
Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species; Abu-Amero KK et al.; Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake . All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol . The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass . Amongst 20 Mycoplasma species tested, M . agalactiae, M . bovis, M . dispar, M . gallisepticum, M . pneumoniae and M . ovipneumoniae oxidised ethanol . Propanol was also oxidised by M . dispar and isopropanol by M . agalactiae, M . bovis and M . ovipneumoniae . Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone . The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.

Photochem Photobiol, 2000 Jan, 71(1), 84 - 9
Isolation and initial characterization of 13(2)-hydroxychlorophyll a induced by cyclohexanedione derivatives in tobacco cell suspension cultures; Wang JM et al.; This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene-5-5-dim ethyl- cyclohexane-1,3-dione (RWH-21), stimulates accumulation of 13(2)-hydroxychlorophyll a in cultured tobacco cells . This was shown based on isolation of 13(2)-hydroxychlorophyll a from pigment extracts of cultured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis . 13(2)-Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH-21 (50 microM) . Analysis of 13(2)-hydroxychlorophyll a formation in tobacco cells indicates that 13(2)-hydroxychlorophyll a is rapidly accumulated within 20 h incubation time both in the dark and light . Although the amount of 13(2)-hydroxychlorophyll a is continuously increased in the dark, the amount of 13(2)-hydroxychlorophyll a decreased remarkably in the light after 20 h incubation . Analysis of 13(2)-hydroxychlorophyll a formation and lipid peroxidation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 13(2)-hydroxychlorophyll a has the potential to cause a photodynamic action in cultured tobacco cells.

Biotechnol Bioeng, 2000 Mar 5, 67(5), 520 - 8
Volume changes of isolated human K562 leukemia cells induced by electric field pulses; Ferret E et al.; Electropermeabilization of immobilized human leukemia K562 cells was studied by measuring changes in cell volume . Such changes reflect mass transfer between the cell and external medium . Electropermeabilization was carried out in an isosmotic water-sorbitol medium with a range of electric field strengths from 500 to 800 V . cm(-1), corresponding to low-energy levels . Electroporation of the K562 cell membrane was found to provoke an inflow of sorbitol and a corresponding osmotic inflow of water and/or an outflow of intracellular solutes due to Fick diffusion . Such flows were found to involve the shrinkage, swelling, or rupture of K562 cells, depending on the characteristics of the electric field and of the physiological state of cells . The behavior of immobilized cells was observed during their exposure to the electric field . The response in immobilized cell volume corresponded with the theoretical pore size and pore opening time, permitting an explanation of the behavior of cell suspensions subject to electrical fields .

J Immunol Methods, 1999 Dec 10, 231(1-2), 65 - 81
Model systems to study the parameters determining the success of phage antibody selections on complex antigens; Mutuberria R et al.; Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens . Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source . However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods . This provides a series of technical and experimental challenges . One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature . To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen" . We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours . Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin . We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers . Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested . Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen . Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described . The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed . The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.

J Autoimmun, 2000 Feb, 14(1), 63 - 78
CD69, HLA-DR and the IL-2R identify persistently activated T cells in psoriasis vulgaris lesional skin: blood and skin comparisons by flow cytometry; Ferenczi K et al.; Many lymphocyte-activation-associated molecules are observed by immunohistochemistry in psoriasis vulgaris lesional skin . Non-T cells in lesional skin also express these molecules . We quantitatively measured the number of T cells expressing cell surface activation-associated molecules (CD69, CD25, CD122, HLA-DR) and co-stimulatory molecules (CD28, CTLA-4, CD80, CD86), including a Type 2 T cell marker (CD30) and CD11b, by flow cytometry of skin and peripheral blood . T cells in single cell suspensions of psoriatic lesional-epidermis-expressed HLA-DR (86%), CD69 (59%), CD25 (55%), CD122 (44%), and CD28 (91%) . Dermal T cells showed similar percentages except for CD69 (17%) . CD69 was found directly in lesional skin biopsies by immunohistochemistry . Both CD4 and CD8 subsets from lesional skin contained large populations of CD25+ cells with a bias towards CD8 activation in the epidermis and towards CD4 activation in the dermis . CD86, CD80, CTLA-4, CD30 and CD11b were expressed by less than 23% of the T cell populations from both the epidermis and dermis . CD30+CD4+ cells were found two-fold over CD8+ T cells . These results show that the majority of lesional lymphocytes are persistently activated . We also found the majority of Type 2 associated markers primarily on the CD4+ epidermal T cell population . Psoriatic blood contained elevated levels of T cells expressing CD25, primarily within the CD8+ subset . Thus the majority of lesional T cells expressed the three primary activation markers, while psoriatic blood T cells were distinguished by an increase in CD25, specifically within the CTL population .

Clin Exp Metastasis, 1999 Jul, 17(5), 377 - 87
Beta1-integrin-mediated dynamic adhesion of colon carcinoma cells to extracellular matrix under laminar flow; Haier J et al.; To resist substantial wall shear stress exerted by blood flow metastasizing colon carcinoma cells have to form adhesive contacts with endothelial cells and subendothelial extracellular matrix (ECM) . At secondary sites tumor cells have to stabilize these initial adhesive interactions to prevent detachment and recirculation . Previously we found that adhesion of colon carcinoma cells to ECM components under static conditions is mediated, in part, by various beta1-integrins . Since other malignant cells possess adhesive properties that are different under static and dynamic conditions, we analyzed human colon carcinoma cell adhesion under flow by decreasing the flow (wall shear stress, WSS) of cell suspensions and allowing cells to interact with collagen-coated surfaces in a laminar flow chamber . HT-29 colon carcinoma cells were used to study wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR) and adhesion stabilization rate (ASR) . DAR was determined after a low flow period using a WSS set at 50% of WSAT . ASR was calculated 60 sec after reestablishment of high WSS . Glass slides were coated with collagen I (C I) or bovine serum albumin (BSA, negative control) . In some experiments cells were pretreated with function-blocking anti-beta1 or nonspecific IgG . Rolling of cells occurred on C I- and BSA-coated surfaces at high WSS . By decreasing WSS cell sticking without definite adhesion was found, and cells stuck to BSA at WSS lower than that found for C I . Further decreasing WSS below WSAT enabled stable cell adhesion to C I, but only a few cells adhered to BSA . ASR was found to be 73% of primarily adherent cells (to C I) . Pretreatment with anti-beta1 did not affect cell rolling but did inhibit cell sticking and adhesion completely, whereas nonspecific IgG was without effect . Activation of PKC using phorbol ester resulted in an increase of adhesive interactions under dynamic and static conditions, whereas its inhibition reduced adhesion . Adhesive interactions of HT-29 colon carcinoma cells with ECM-coated surfaces under laminar flow conditions occurred in various steps: (1) rolling, (2) sticking or initial adhesion, and (3) stabilization of adhesion . Under shear flow rolling of tumor cells on ECM-coated surfaces appeared to be mediated mainly by physical/mechanical and nonspecific surface-cell membrane interactions, whereas stabilized adhesion to ECM was specifically mediated by beta1-integrin binding to ECM components . PKC seems to be involved in the regulation of adhesion stabilization under static and flow conditions.

Neurotoxicol Teratol, 2000 Jan-Feb, 22(1), 41 - 53
Intrahippocampal cholinergic-rich transplants restore lead-induced deficits: a preliminary study in rats; Adhami VM et al.; In the present study restorative potential of fetal cholinergic rich cell suspensions in ameliorating cognitive deficits in rats perinatally exposed to lead was studied . Lactating dams with 1-day old litters were given 0.2% (w/v) lead acetate in drinking water throughout lactation from postnatal day (PND) 1 to PND21 at the end of which the treatment was stopped and the animals were weaned . On PND42 lead exposed rats were given bilateral, intrahippocampal, cholinergic rich fetal neural transplants (approximately 60,000 cells per site) and subsequently assessed 3 and 6 months posttransplantation . Control animals (Sham operated and transplanted) were also run in parallel . Lead exposed rats exhibited a decreased learning ability and locomotor activity . A significant decrease in the levels of acetylcholinesterase and sodium potassium ATPase Na+,K+-ATPase activity was observed in hippocampal region of lead exposed rats . The levels of lead were increased by fivefold in the hippocampal region of lead exposed rats . Transplantation showed marginal improvement in the above impairments at 3 months which were more marked at 6 months . Lead levels at 6 months were not significantly higher in lead exposed rats as compared with the control . Results confirm previous findings that fetal neural transplants help in restoring the lost functional deficits and demonstrate their restorative potential in case of lead induced deficits.

Exp Hematol, 1999 Dec, 27(12), 1768 - 75
Characterization of germinal center dendritic cells in follicular lymphoma; Renard N et al.; A subset of dendritic cells called germinal center dendritic cells (GCDC) has recently been described inside germinal center from reactive lymphoid organs . We investigated this newly recognized population in follicular lymphoma (FL), which is considered to be the pathologic counterpart of germinal center B cells . Immunohistochemistry analysis with a panel of antibodies demonstrated the presence of a cell population with the peculiar GCDC phenotype in FL biopsies and a similar localization of these cells inside tumoral and reactive follicles . Therefore, we analyzed the relationships between GCDC and the other cell subsets of the tumor follicles . Some of CD4+ and CD8+ T lymphocytes present inside the follicle were found to be in close association with GCDC, suggesting a potential implication of GCDC in their activation . In addition, the distribution of GCDC inside FL and reactive follicles did not appear disrupted, in contrast to follicular dendritic cells, the other follicle dendritic cell type . Finally, we demonstrated that GCDC could be detected from FL lymph node cell suspension by flow cytometry . Taken together, these results indicate that FL development is not associated with a disappearance of GCDC or with a lack of physical interactions between GCDC and T cells inside the follicles . In addition, the fact that GCDC can be observed in FL samples by flow cytometry should allow their purification to further study their putative role in FL development and maintenance.

Plant Cell, 2000 Jan, 12(1), 97 - 110
The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development; Vernoux T et al.; Activation of cell division in the root apical meristem after germination is essential for postembryonic root development . Arabidopsis plants homozygous for a mutation in the ROOT MERISTEMLESS1 (RML1) gene are unable to establish an active postembryonic meristem in the root apex . This mutation abolishes cell division in the root but not in the shoot . We report the molecular cloning of the RML1 gene, which encodes the first enzyme of glutathione (GSH) biosynthesis, gamma-glutamylcysteine synthetase, and which is allelic to CADMIUM SENSITIVE2 . The phenotype of the rml1 mutant, which was also evident in the roots of wild-type Arabidopsis and tobacco treated with an inhibitor of GSH biosynthesis, could be relieved by applying GSH to rml1 seedlings . By using a synchronized tobacco cell suspension culture, we showed that the G(1)-to-S phase transition requires an adequate level of GSH . These observations suggest the existence of a GSH-dependent developmental pathway essential for initiation and maintenance of cell division during postembryonic root development.

Plant Cell, 2000 Jan, 12(1), 65 - 80
Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P; Bruce W et al.; To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors . BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site . Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol . The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment . Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr . The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected . Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns . The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected . GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.

Exp Neurol, 1999 Nov, 160(1), 88 - 98
Apoptosis in primary cultures of E14 rat ventral mesencephala: time course of dopaminergic cell death and implications for neural transplantation; Branton RL et al.; Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum . However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery . A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear . Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival . Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media . After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically . Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h . Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons . There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h . We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.

Z Naturforsch {C}, 1999 Nov, 54(11), 993 - 5
Activation of hematoporphyrin in alternating magnetic field: possible implications for cancer treatment; Babincova M et al.; A new mechanism of cell damage by alternating magnetic field with hematoporphyrin is described . C6 glioblastoma cell suspensions were exposed to an alternating magnetic field with frequency 180 kHz up to 60 min in the presence of hematoporphyrin in H2O and in D2O . The results presented suggest that an alternating magnetic field is able to activate hematoporphyrin, and this method may be a basis for cancer treatment.

Clin Orthop, 1999 Aug, (365), 149 - 62
Histologic analysis of tissue after failed cartilage repair procedures; Nehrer S et al.; This study evaluated the composition of reparative tissue retrieved during revision surgery from full thickness chondral defects in 18 patients in whom abrasion arthroplasty (n = 12), grafting of perichondrial flaps (n = 4), and periosteal patching augmented by autologous chondrocyte implantation in cell suspension (n = 6) failed to provide lasting relief of symptoms . The defects were graded by gross appearance, and all of the tissue filling the defect was retrieved . Histologic evaluation included histomorphometric analysis of the percentage of selected tissue types in cross sections . Immunohistochemistry was performed using antibodies to Types I, II, and X collagen . The histologic appearance of material retrieved after abrasion arthroplasty was that of fibrous, spongiform tissue comprising Type I collagen in 22% +/- 9% (mean +/- standard error of the mean) of the cross sectional area, and degenerating hyaline tissue (30% +/- 10%) and fibrocartilage (28% +/- 7%) with positive Type II collagen staining . Three of four specimens obtained after implantation of perichondrium failed as a result of bone formation that was found in 19% +/- 6% of the cross sectional area, including areas staining positive for Type X collagen, as an indicator for hypertrophic chondrocytes . Revision after autologous chondrocyte implantation was associated with partial displacement of the periosteal graft from the defect site because of insufficient ongrowth or early suture failure . When the graft edge displaced, repair tissue was fibrous (55% +/- 11%), whereas graft tissue attached to subchondral bone displayed hyaline tissue (to 6%) and fibrocartilage (to 12%) comprising Type II collagen at 3 months after surgery . Evaluation of retrieved repair tissue after selected cartilage repair procedures revealed distinctive histologic features reflecting the mechanisms of failure.

Breast Cancer Res Treat, 1999 Oct, 57(3), 271 - 5
Antimetastatic effect of desmopressin in a mouse mammary tumor model; Alonso DF et al.; We have investigated the effects of desmopressin (DDAVP), a synthetic analog of the natural hormone vasopressin, on experimental lung colonization of mammary tumor cells using a syngeneic BALB/c mouse model . Coinjection of DDAVP (1-2 microg/kg body weight) at the time of i.v . inoculation of F3II carcinoma cells or LM3 adenocarcinoma cells significantly inhibited the formation of experimental lung metastases . In both cases, the number of pulmonary nodules was reduced about 70% . Inhibition of metastasis was also obtained with i.v . administration of DDAVP 24 h after tumor cell inoculation . Interestingly, the inhibition of lung metastasis was not due to direct cytotoxic effects of DDAVP on mammary tumor cells . The in vitro formation of multicellular aggregates in the presence of citrated plasma from control and DDAVP-treated mice was also examined . Control plasma rapidly induced a significant tumor cell aggregation . In contrast, in the presence of plasma from DDAVP-treated mice, tumor cells remained as a single cell suspension . DDAVP may help to dissolve the protective fibrin shield of circulating tumor cells . Our data suggest, for the first time, that adjuvant DDAVP therapy may impair successful implantation of circulating mammary tumor cells.

Ann Thorac Surg, 1999 Dec, 68(6), 2074 - 80; discussion 2080-1
Autologous heart cell transplantation improves cardiac function after myocardial injury; Sakai T et al.; BACKGROUND: Fetal ventricular cardiomyocyte transplantation into a cardiac scar improved ventricular function, but these cells were eventually eliminated by rejection . We therefore examined the feasibility of autologous adult heart cell transplantation . METHODS: A transmural scar was produced in the left ventricular free wall of adult rats by cryoinjury . The left atrial appendage was harvested, and the atrial heart cells were cultured and their number expanded ex vivo . Three weeks after cryoinjury, either a cell suspension (2 x 10(6) cells, n = 12 rats, transplant group) or culture medium (n = 10 rats, control group) was injected into the scar . Rats having a sham operation (n = 5) did not undergo cryoinjury or transplantation with cells or culture medium . RESULTS: Five weeks after injection, ventricular function was evaluated in a Langendorff preparation, measuring systolic, diastolic, and developed pressures over a range of intraventricular balloon volumes . Systolic and developed pressures were greater in the transplant group than in the control group (p = 0.0001) . Rats with a sham operation had the greatest systolic, diastolic, and developed pressures (p = 0.0001) . Histologic studies demonstrated survival of the transplanted heart cells within the scar . The area of the scar was smaller (p = 0.0003) and its thickness greater (p = 0.0003) in rats in the transplant group . Left ventricular chamber volume was smaller in the transplant group (p = 0.043) . CONCLUSIONS: Transplantation of autologous cultured adult atrial heart cells limited scar thinning and dilatation and improved myocardial function compared with results in control hearts . This technique may lead to a novel therapy to prevent scar expansion after a myocardial infarction and prevent the development of congestive heart failure.

J Neurosurg, 2000 Jan, 92(1), 127 - 31
Reduced xenograft rejection in rat striatum after pretransplant photodynamic therapy of murine neural xenografts; Honey CR et al.; OBJECT: The goal of this study was to develop a method of reducing neural xenograft rejection by pretreating the graft with photodynamic therapy (PDT) . METHODS: Xenograft cell suspensions were prepared from fetal mouse mesencephalon, after which they were incubated for 30 minutes with various concentrations of a photosensitizer, verteporfin for injection, and light exposure . The xenograft cell suspensions were injected into the dopamine-depleted striata of 40 hemiparkinsonian rats assigned to different treatment groups . Four weeks after transplantation, xenograft function (determined by methamphetamine-induced rotation) and survival (determined by immunohistochemical staining for murine neurons) were compared . Group 1 animals (xenografts pretreated with 25 ng/ml verteporfin) and Group 3 animals (no verteporfin pretreatment, but daily administration of cyclosporin A) had significantly better xenograft survival and function compared with control animals (no pretreatment with verteporfin) . Group 2 animals (xenografts pretreated with 250 ng/ml verteporfin) had no significant improvement . CONCLUSIONS: This work demonstrates improved neural xenograft survival and function when using pretransplant PDT of the graft in a rodent model . The potential benefits of this new therapy are its convenience (one pretransplant treatment) and its compatibility with host immunosuppression.

IEEE Trans Biomed Eng, 1999 Dec, 46(12), 1413 - 25
Rapid electromagnetic warming of cells and tissues; Robinson MP et al.; We describe a system for thawing frozen cell suspensions and tissues by electromagnetic absorption . A 25-ml sample is heated in a cylindrical resonant cavity, which is excited in three modes all close to 434 MHz . Maximum warming rates are over 10 degrees C/s (600 C/min), and a frozen sample may be brought from -65 degrees C to room temperature in < 30 s, with final spatial differences of < 20 degrees C . Samples may be frozen externally, or cooled within the cavity at typically 1 degree C/min . We have also used the resonant cavity to measure the permittivity and conductivity of the sample at temperatures from -83 degrees C to +8 degrees C . By measuring the heat capacity of the sample, we have calculated the power deposited in it as a function of its temperature . The system is currently being used to investigate the effect of warming rate on cell survival.

Tissue Eng, 1999 Dec, 5(6), 563 - 72
Cultured epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full-thickness wounds; Voigt M et al.; Research efforts to modify cultured autologous skin transplants for large full-thickness burn wounds and in chronic ulcers have shifted from multilayered differentiated grafts ("sheet" grafts) toward smaller units of basal undifferentiated single cell suspensions in a transport medium and subconfluently covered static carriers . It has been shown that wounds transplanted with single cell suspensions reconstitute the epidermis . However, this technique requires the detachment of the keratinocytes from the culture flasks by enzymatic digestion-digestion that might alter the anchoring proteins of the cells . A new approach might be to circumvent the enzymatic digestion to harvest the keratinocytes . This study reports a technique to culture epidermal cells on spherical microcarriers as a suspension culture and transport vehicle . The spherical microcarrier consists of a 100-microm-diameter collagen-coated dextran carrier (Cytodex 3 Pharmacia) and has been used previously for enzyme production commercially . With this new approach, we seeded the human keratinocytes in a spinner-like system onto microspheres and transplanted these micrografts onto full-thickness wounds on the back of nude mice . After 14 days, we showed a reconstituted epithelium that was multilayered and keratinized compared to control wounds . We believe that this is the first step of a new approach to increase the cell yield for seeding without altering the anchoring proteins by enzymatic steps, leading to a superior transplantation method for keratinocytes.

Biofactors, 1999, 10(2-3), 179 - 85
Changes in several functions of murine peritoneal macrophages by N-acetylcysteine and thioproline ingestion . Comparative effect between two strains of mice; Blanco B et al.; The administration of the thiol compounds, N-acetylcysteine (NAC) and in particular thioproline (thiazolidine-4-carboxylic acid) at 0.1% w/w concentration in the diet, improves lymphocyte functions in old female Swiss mice, as has been shown in our previous studies . In the present work, adult mice from two different strains, namely BALB/c (an inbred strain) and OF1-Swiss (noninbred strain), were fed a diet supplemented with the above dose of each thiol compound jointly for five weeks . At 28 weeks of age, peritoneal cell suspensions were obtained and different steps of the phagocytic process, the most representative activity of macrophages, as well as interleukin-1beta (IL-1beta) production, were studied . Thus, adherence to substrate, mobility directed to a chemoattractant gradient (chemotaxis), ingestion of inert particles and superoxide anion production were analysed . The results show that diet supplementation with NAC plus thioproline increased all macrophage functions studied with the exception of superoxide anion production, which was decreased . These effects were more evident in macrophages from Swiss mice, whereas in BALB/c mice the stimulation of phagocytosis and IL-1beta production was lower and no differences were seen after treatment in adherence and superoxide anion production . These data suggest that immune function can be improved in adult mice by administration of the above thiol compounds, especially in the noninbred strain of OF1-Swiss mice.

J Biol Chem, 1999 Dec 31, 274(53), 37915 - 22
Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors; Vollmer JY et al.; A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells . On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer . This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses . On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence . Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area . Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application . Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

Int J Pharm, 2000 Jan 5, 193(2), 219 - 26
Safety assessment of selected cyclodextrins - effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis; Uchenna Agu R et al.; The objective of this study was to assess the cilio-inhibitory effect of a series of cyclodextrins using a human cell suspension culture system exhibiting in vitro ciliogenesis . Enzymatically released human nasal epithelial cells were cultured as sequential monolayer-suspension culture showing in vitro ciliogenesis . Ciliary beat frequency (CBF) was determined by computerized microscope photometry . Among the cyclodextrins investigated (gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, anionic-beta-cyclodextrin polymer, dimethyl-beta-cyclodextrin and alpha-cyclodextrin), it was shown that after 30 min of exposure, gamma-cyclodextrin (10% w/v), hydroxypropyl-beta-cyclodextrin (10.0% w/v) and anionic-beta-CD polymer (8.0% w/v) were not significantly cilio-inhibitory (P0.05) . Similarly, CBF remained stable upon cell exposure to alpha-cyclodextrin (2.0% w/v) and dimethyl-beta-cyclodextrin (1.0% w/v) . However, higher concentrations of alpha-cyclodextrin and dimethyl-beta-cyclodextrin resulted in mild to severe cilio-inhibition after 45 min of exposure . The effect of alpha-cyclodextrin (5.0% w/v; 54+/-4% cilio-inhibition) was partially reversible while dimethyl-beta-cyclodextrin (10% w/v; 36+/-4% cilio-inhibition) was irreversible . The cilio-inhibition observed in this model was lower than reported for chicken trachea model . Given the fact that (1) irreversible cilio-inhibition observed in this study occurred only at concentrations exceeding those used in pharmaceutical formulations and/or at an unusual exposure time (45 min) and that (2) in an in vivo situation, dilution and mucociliary clearance contribute to further decrease in local concentrations of the applied compound, the results of this study confirm the safety of the cyclodextrins investigated as nasal absorption enhancers.

Neuroreport, 1999 Nov 8, 10(16), 3347 - 51
Neuronal death in nigral grafts in the absence of poly (ADP-ribose) polymerase activation; Kaminski Schierle GS et al.; The exact causes of the extensive cell death in nigral transplants are still unknown . Since poly-(ADP-ribose) polymerase (PARP) overactivation has been implicated in neuronal death, we examined the effects of PARP on the survival of nigral grafts by using donor tissue from PARP knock-out or wild-type mice . Eight hours after preparation of the nigral cell suspension, cell damage was quantified by measurement of lactate dehydrogenase release, DNA fragmentation and caspase activation . At this stage, PARP deletion had no protective effect . Moreover, neither the survival of transplanted dopaminergic neurons, nor the functional recovery of hemiparkinsonian graft recipients were improved by the absence of PARP . We conclude that cell death in embryonic nigral grafts is not affected by the absence of PARP activation.

Lipids, 1999 Nov, 34(11), 1143 - 9
Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape; Byers SD et al.; Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L . cv . Jet Neuf) . At a concentration of 25 mM, MgSO4 and MgCl2 stimulated microsomal DGAT 25- and 10-fold, respectively . ATP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg2+ concentrations . Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate . sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect . DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction . This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.

Biochim Biophys Acta, 2000 Jan 3, 1483(1), 119 - 31
The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats; Retterstol K et al.; The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells . The amounts of {(14)C}22- and 24-carbon metabolites were measured by HPLC . The conversion of {1-(14)C}20:5n-3 to {3-(14)C}22:6n-3 was more efficient than that of {1-(14)C}20:4n-6 to {3-(14)C}22:5n-6 . At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM) . The conversion of {1-(14)C}22:5n-3 to {1-(14)C}22:6n-3 was 1.7 times more efficient than that of {1-(14)C}22:4n-6 to {1-(14)C}22:5n-6 using a low, but almost equally efficient using a high substrate concentration . When unlabelled 20:5n-3 was added to a cell suspension incubated with {1-(14)C}20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with {1-(14)C}22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of {1-(14)C}20:4n-6 or {1-(14)C}22:4n-6 to {(14)C}22:5n-6 . In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of {1-(14)C}20:5n-3 and {1-(14)C}22:5n-3 to {(14)C}22:6n-3 . The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids . In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.

Am J Physiol, 1999 Dec, 277(6 Pt 2), R1741 - 8
PGE2 suppresses mitogen-induced Ca2+ mobilization in T cells; Choudhry MA et al.; PGE2-mediated suppression of T cell proliferation during sepsis could result from altered Ca2+ signaling . The present study evaluated the effects of PGE2 on Ca2+ release from intracellular stores and its influx through the plasma membrane in splenic T cells from Sprague-Dawley rats . Intracellular Ca2+ concentration ({Ca2+}i) responses in individual T cells were assessed using the Ca2+ imaging technique, and the release of Ca2+ from intracellular stores and Ca2+ influx were spectrofluorometrically quantified in T cell suspensions . Under unstimulated conditions, nearly 85% of T cells exhibited {Ca2+}i </=50 nM . After stimulation with concanavalin A (Con A), an increase in {Ca2+}i was recorded in approximately 60% of the cells . The pretreatment of T cells with PGE2 had no apparent effect on {Ca2+}i in resting cells; it significantly suppressed the Con A-induced increase in {Ca2+}i in all of the Con A-responsive cells . Ca2+ release from the intracellular stores contributed to the early spike in {Ca2+}i, and the late phase of elevation in {Ca2+}i was dependent on Ca2+ influx through the plasma membrane . Our data suggest that PGE(2) causes an overall suppression of the Con A-induced {Ca2+}i elevation in T cells via inhibiting both Ca2+ influx and its release from the intracellular stores.

J Struct Biol, 1999 Dec 15, 128(2), 187 - 99
Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells; Hardt M et al.; We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX) . Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding . The quenched-flow used allowed analyses, during stimulation, in the subsecond time range . Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable . We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation . These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter) . We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual inverted question markCa . Calibration curves showed a linear relationship and a detection limit of inverted question markCa = 2 mM, while inverted question markCa in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells . Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes . With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores .

Cryobiology, 1999 Nov, 39(3), 228 - 35
Freeze-drying of red blood cells: how useful are freeze/thaw experiments for optimization of the cooling rate?
Rindler V, Heschel I, Rau G.
A red blood cell suspension, prepared according to a high-yield HES cryopreservation protocol, was frozen at selected cooling rates of 50, 220, 1250, 4200, and 13,500 K/min . After either thawing or vacuum-drying, the cell recovery was determined using a modified saline stability test . As expected, the recovery of thawed samples followed the theory of Mazur's two-factor hypothesis . The best result was found at a cooling rate of 220 K/min . In contrast, the recovery of freeze-dried and rehydrated samples was very poor at that rate, but maximal at 4200 K/min where thawing caused almost complete hemolysis . This discrepancy is attributed to different damaging mechanisms involved with the respective sample processing subsequent to freezing . While thawing leads to increased devitrification and recrystallization at supraoptimal cooling rates for cryopreservation, the resultant almost vitreous sample structure seems to be advantageous for vacuum-drying . It can be concluded that freeze/thaw experiments are not sufficient for optimization of the cooling rate for freeze-drying .

Zentralbl Veterinarmed A, 1999 Oct, 46(8), 459 - 64
Flow cytometry analysis of milk and peripheral blood cells from goats during lactation; Winnicka A et al.; Cells from goat's milk and peripheral blood taken during the lactation period were analysed by flow cytometry . The investigated cells were populations of leucocytes, lymphocyte subpopulations (T, T-helper, T-cytotoxic, B, WC1-N2) and all MHC class II positive cells . Labelling of cells was performed on whole blood and milk cell suspensions . Statistically significant differences were found between percentages of B and WC1-N2 lymphocytes and MHC II positive cells from peripheral blood during the lactation period and all of examined milk cells during the same time.

J Agric Food Chem, 1999 Aug, 47(8), 3292 - 6
Compartive study of volatile components and fatty acids of plants and in vitro cultures of parsley (Petroselinum crispum (Mill) nym ex hill); Lopez MG et al.; Volatile compounds from plants, callus tissue cultures, and cell suspensions of parsley (Petroselinum crispum) were captured during the growth cycle using a dynamic headspace extraction and were identified by gas chromatography-mass spectrometry . Parsley plants were found to produce mainly monoterpenes, and the compound of major abundance was p-1,3,8-menthatriene, followed by beta-phellandrene and apiole . Callus cultures and cell suspensions produced aldehydes (nonanal and decanal) that were also detected in parsley plant . The former also produced limonene, acetophenone, and benzotiazol; these were not observed in the plants . The production of volatiles in plants, callus tissue, and cell suspensions was found to be time-dependent . Free and bound fatty acids were also monitored by an in situ method . Palmitic (C16:0) and stearic (C18:0) acids were the most abundant fatty acids in all materials; however, higher levels were found in plants . On the other hand, the unsaturated C16:1 and C16:3 were not detected in the in vitro cultures.

Eur J Neurosci, 1999 Dec, 11(12), 4341 - 8
Dopamine cells in nigral grafts differentiate prior to implantation; Sinclair SR et al.; The yield of surviving dopamine cells in nigral grafts is typically low . It is unclear whether the dopamine neurons that do survive are postmitotic at the time of implantation, or are precursor cells that differentiate into dopamine neurons following transplantation in the host brain . We have therefore compared the survival of dopamine neurons in grafts that have been labelled with BrdU at different times prior to or following implantation in order to identify those cells that undergo final cell division at each stage of the procedure . Seven groups of rats were prepared with unilateral nigrostriatal lesions . Three groups received nigral grafts derived from E14 embryos labelled with BrdU in utero on either E12, E13 or E14 days of embryonic age (the E14 injection made 2 h prior to preparation of the graft cell suspension) . Three further groups received nigral grafts from untreated E14 embryos, and then dividing cells within the grafts were labelled by injection of BrdU into the host lateral ventricle, 2 h, 1 day or 2 days after implantation (equivalent to E14, E15 and E16 days of embryonic age) . The control group received standard (unlabelled) E14 grafts . Five weeks after the transplantation surgery, the host brains were processed using double immunohistochemical techniques to detect tyrosine hydroxylase (TH)-positive neurons which had incorporated BrdU . In the grafts labelled with BrdU prior to implantation, there was an increasing proportion of double-labelled cells (out of the total TH-positive cells surviving in the grafts) with birth dates on E12, E13 and E14 (1%, 12% and 10% per day, respectively) . By contrast, grafts labelled following implantation, although containing many dividing neurons, had very few of these BrdU-labelled cells expressing a dopaminergic phenotype; < 1% surviving TH-positive cells were double-labelled from the 2 h post-transplant injection, and < 0.1% from each subsequent injection . This suggests not only that the great majority of TH-positive neurons in nigral grafts were already differentiated at the time of implantation, but also that transplantation of E14 ventral mesencephalic tissue either kills dopaminergic precursors or (more likely in our opinion) prevents their differentiation into a dopaminergic phenotype . Precursor cells that would differentiate into dopaminergic neurons beyond E14 if left in situ in the intact ventral mesencephalon do not readily differentiate into mature dopamine neurons following transplantation . If we are to enhance yields of functional dopamine-rich transplants, then we must identify strategies both to protect predifferentiated dopamine neurons in the grafts and to promote differentiation of a dopaminergic phenotype in precursor cells that continue to divide within the grafts following transplantation into an adult host environment.

Plant Physiol, 1999 Dec, 121(4), 1299 - 308
N-Acylethanolamines in signal transduction of elicitor perception . Attenuation Of alkalinization response and activation of defense gene expression
Tripathy S, Venables BJ, Chapman KD.
In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g . N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D . Chapman, S . Tripathy, B . Venables, A.D . Desouza {1998} Plant Physiol 116: 1163-1168) . In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors . Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants . In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner . Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco . NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants . Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.

Planta, 1999 Nov, 210(1), 157 - 64
Alfalfa and tobacco cells react differently to chitin oligosaccharides and sinorhizobium meliloti nodulation factors
Baier R, Schiene K, Kohring B, Flaschel E, Niehaus K.
Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity . The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L . ) cell-suspension cultures were treated with yeast elicitors . Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst . Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst . In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst.

Planta, 1999 Nov, 210(1), 150 - 6
Biosynthetic origin and longevity in vivo of alpha-d-mannopyranosyl-(1 --> 4)-alpha-d-glucuronopyranosyl-(1 --> 2)-myo-inositol, an unusual extracellular oligosaccharide produced by cultured rose cells
Smith CK, Fry SC.
A non-reducing trisaccharide, alpha-D-mannopyranosyl-(1 --> 4)-alpha-D-glucuronopyranosyl-(1 --> 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of 'Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth . This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.) . When added to cultured Rosa cells, {(14)C}MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly . When D-{6-(14)C}glucuronic acid was fed to cultured Rosa cells, extracellular {(14)C}MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides . Furthermore, {(14)C}MGI continued to accumulate in the medium for at least 20 h after the accumulation of (14)C-polymers had ceased . These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate . Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid) . No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells . We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI.

Vet Microbiol, 1999 Oct, 70(1-2), 21 - 31
Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls; Gradil CM et al.; Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls) . Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing . The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays . Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58 . BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65 . PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98 . Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful . Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations . Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR . No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.

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