Cell suspensions

Allergy, 2004 Apr, 59(4), 428 - 35
Endobronchial eosinophils preferentially stimulate T helper cell type 2 responses; Shi HZ et al.; BACKGROUND: Antigen-loaded eosinophils instilled intratracheally into mice were capable of migrating into local lymph nodes and localize to the T cell-rich paracortical zones where they stimulated antigen-specific proliferation of CD4+ T cells . The aim of the present study was to evaluate whether eosinophils within the tracheobronchial lumen can stimulate Th2 cell expansion by presenting antigen both in vitro and in vivo . METHODS: Airway eosinophils were recovered from ovalbumin-sensitized and -challenged BALB/c mice, these eosinophils were then co-cultured with sensitized CD4+ cells in the absence or presence of anti-CD80 or/and -CD86 monoclonal antibodies . Airway eosinophils were instilled into the trachea of sensitized mice . At 3 days thereafter, the draining paratracheal lymph nodes were removed and teased into cell suspensions for culture . Cell-free culture supernatants were collected for detection of cytokines . RESULTS: Our data showed that airway eosinophils functioned as CD80- and CD86-dependent antigen-presenting cells (APCs) to stimulate sensitized CD4+ lymphocytes to produce interleukin (IL)-4, IL-5, and IL-13, but not interferon (IFN)-gamma in in vitro assay . When instilled intratracheally in sensitized recipient mice, airway eosinophils migrated into draining paratracheal lymph nodes primed Th2 cells in vivo for IL-4, IL-5, and IL-13, but not IFN-gamma, production during the in vitro culture that was also CD80- and CD86-dependent . CONCLUSION: Eosinophils within the lumina of airways could process inhaled antigen function in vitro and in vivo as APCs to promote expansion of Th2 cells . This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate immune responses by amplifying Th2 cell responses.

Proteomics, 2004 Mar, 4(3), 720 - 7
Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv . Bright Yellow-2 cell suspension culture; Laukens K et al.; Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv . Bright Yellow-2 (tobacco BY-2) . The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells . We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture . A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching . These data were integrated in a database, which can be accessed at On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries . Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases . Comprehensive search functions are implemented . Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS . This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.

Int Immunopharmacol, 2004 Feb, 4(2), 289 - 98
Induction of MLR-Bf and protection of fetal loss: a current double blind randomized trial of paternal lymphocyte immunization for women with recurrent spontaneous abortion; Pandey MK et al.; The present study was conducted to evaluate the efficacy of paternal lymphocyte (PL) immunotherapy and its relation with the development of mixed lymphocyte reaction blocking antibodies (MLR-Bf) and the success of pregnancy outcome in women with recurrent spontaneous abortion (RSA) . A total of 124 women with unknown causes of abortions was registered for immunotherapy under double blind randomized trial by using the list of computer-generated numbers . Each 5 x 10(6) autologous lymphocyte (AL), third party lymphocyte (TPL) and PL was dissolved separately in 1 ml of sterile normal saline (NS) . Each 1 ml of cell suspension and neat NS was injected in women with RSA through intramuscular (250 microl), intradermal (250 microl), subcutaneous (250 microl) and intravenous (250 microl) routes . All women participants with RSA received six identical immunizations at the regular interval of 4 weeks, and were then screened for the development of MLR-Bf after the completion of immunization course, and also at the first, second and third trimesters (12th, 24th and 36th weeks) of pregnancy . However, nonimmunized MLR-Bf positive women with RSA did not receive any kind of therapy (NT) and were used as one of the control group in the present study . We have observed that PL-immunized women with RSA showed a significantly increased level of MLR-Bf (>30) and pregnancy success (84%) as compared to those women with RSA who received either AL (33%), TPL (31%), NS (25%) or those who did not receive any kind of treatment (NT, 44%; P<0.001) . Our results indicated the importance of immunotherapy with PL in women with RSA and also showed that MLR-Bf can be considered as one of the important factors for pregnancy improvement.

J Agric Food Chem, 2004 Mar 10, 52(5), 1138 - 45
Biosynthesis and characterization of 14C-enriched flavonoid fractions from plant cell suspension cultures; Yousef GG et al.; A range of radiolabeled anthocyanins, proanthocyanidins, and other flavonoids were accumulated by cell suspension cultures of two plant species, ohelo (Vaccinium pahalae) and grape (a Vitis hybrid, Bailey Alicant A), after providing uniformly labeled {(14)C}sucrose to the medium . Approximately 15% of administered label was recovered in a series of flavonoid-rich fractions varying in composition . Anthocyanins, and monomers to oligomers of proanthocyanidins, were labeled effectively and characterized from both species . Most of the proanthocyanidin oligomers were based on the flavan-3-ols (+)-catechin and (-)-epicatechin . Cyanidin and peonidin glycosides were the dominant forms of anthocyanins in both species . Whereas the predominant form of flavonoids identified from ohelo cell cultures was proanthocyanidins, grape cell cultures produced mostly anthocyanins . The labeled phytochemicals were produced for use in subsequent in vivo animal feeding studies to gauge their bioavailability and accumulation in target organs.

Shanghai Kou Qiang Yi Xue, 2001 Jun, 10(2), 138 - 41
{Effects of millimeter wave combined with gamma-ray radiation on human tongue squamous cell carcinoma cell}; Tang YJ et al.; OBJECTIVE: To observe the effects of millimeter wave combined with (60)Co gamma-ray radiation on human tongue squamous cell carcinoma cell(Tca8113) . METHODS: Using mm-wave combined with (60)Co gamma-ray to irradiate Tca8113 cell suspensions . The colony forming inhibition efficiency was observed three weeks later . 24 hours after radiation, the samples were observed under electron microscope . RESULTS: The study showed that the radiation could result in significant decrease of the colony forming efficiency (P<0.001) . The inhibition efficiency was higher when the samples were exposed to high power density mm-wave or for a long duration (P<0.05 or P<0.01) . The inhibiting effect in combined groups was more obvious than in singly radiated group (P<0.001 or P<0.05) . There was no difference between 'R+HL' group and 'HL+R' group (P>0.05) . Electron microscopy showed the cells' suprastructures had some injuries and regressive changes . And more serious changes existed in 'H' group . CONCLUSION: It could be concluded that mm-wave radiation could efficiently inhibit the colony forming capability of Tca8113 cell . And mm-wave radiation could lead to morphological changes of exterior or interior ultrastructures of Tca8113 cell.

Mol Reprod Dev, 2004 Apr, 67(4), 478 - 86
In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation; Ritta MN et al.; Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract . Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena . Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function . The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved . GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml) . Scatchard analysis of {(3)H}-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.) . {(3)H}-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine . Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay) . We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin . It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R .

Mol Reprod Dev, 2004 Apr, 67(4), 446 - 57
Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates; Devireddy RV et al.; Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes . In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa . Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol . Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume . By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined . The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N . s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg){cpa} = 0.90 x 10(-15) m(3)/N . s (0.005 microm/min-atm) and E(Lp){cpa} = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99) . The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice . The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates . This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa .

Biotechnol Bioeng, 2004 Mar 30, 85(7), 714 - 21
Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures; Wu J et al.; This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism . Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel) . The culture was exposed to low-frequency US for a short period of time (2 min) . At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability . The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS) . Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production . Inhibition of the ROS production also suppressed the US-induced JA accumulation . These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T . chinensis cells by low-energy US .

Hum Reprod, 2004 Apr, 19(4), 948 - 53 Epub 2004 Feb 27.
Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells; Frederickx V et al.; BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment . In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol . METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05) . We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference . In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions . In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions . In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively . CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions . Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.

Bioelectrochemistry, 2004 Apr, 62(1), 95 - 8
Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans; Lambreva M et al.; The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes) . There are two possibilities, either:(A)irradiation starts with the electropulse-showed with easily penetrating thiopyronin-yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells . In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill . This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g . chromopeptides) to transport them through cell walls and membranes into cells and tissues . The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.

Zhonghua Yi Xue Za Zhi, 2004 Jan 2, 84(1), 38 - 42
{Migration and differentiation of exogenous rat mesenchymal stem cells engrafted into normal and injured hearts of rats}; Niu LL et al.; OBJECTIVE: To investigate the effects of different microcircumstances on the migration and differentiation of grafted rat mesenchymal stem cells (rMSC) in host myocardium and the feasibility of treatment of myocardial infarction by exogenous adult stem cells . METHODS: rMSC were isolated from the femurs and tibiae of a male Wistar rat and then purified, made into cell suspension, and labeled with DAPI . 35 female Wistar rat were divided randomly into four groups: acute myocardial infarction control group (AMI group, n = 10, the descending anterior branch of left coronary artery was ligated), acute myocardial infarction + rMSC transplantation group (AMI + rMSC group, n = 10, 1 - 3 hours after the ligation DAPI-labeled rMSC were injected into the peri-infarct tissues), normal heart + rMSC transplantation group (normal heart + MSC group, n = 10, DAPI-labeled rMSC were injected into the corresponding myocardium), and mono-nuclear cells transplantation group (AMI + MNCS, n = 5 DAPI-labeled mononuclear cells were injected into he periinfarct tissues) . Ten weeks after the implantation, the rats were killed and their hearts were harvested . Immunohistochemistry was used to examine the troponin, GATA-4 and connexin-43 . RESULTS: No lymphocyte proliferation and immonologic rejection were seen in the cardiac tissues of the rats implanted with rMSC . DAPI-labeled rMSC with blue nuclei were distributed extensively in the myocardium of the AMI + rMSC group, ovoid in shape and arranged in parallel with the cardiac muscle fibers, and were distributed sporadically like islands in the myocardium of the normal heart + rMSC group, irregular in shape and not arranged in parallel with the cardiac muscle fibers . No blue nucleus was seen in the cardiac tissues of the hearts implanted with DAPI-labeled mononuclear cells . Troponin and GATA4 were positive immunohistochemically in the implanted rMSC with blue nuclei and the host cardiac muscle cells of the AMI group and AMI + rMSC group, however, were negative in the implanted rMSC with blue nuclei and normal cardiac muscle cells of the normal heart + rMSC group . CONCLUSION: Purified rMSC are immunologically tolerable and can be used as donor cells for exogenous cells therapy . Capable of surviving and homing in both in normal and injured hearts, exogenous rMSC migrate and differentiate into cardiac muscle cell-like cells in myocardium with infarction, however, not in normal heart.

Anticancer Res, 2003 Nov-Dec, 23(6C), 4871 - 5
In vitro transfection of human bladder cancer cells by acoustic energy; Schaaf A et al.; BACKGROUND: The objective of this study was to examine and quantify the shock-wave-induced transfection of human bladder carcinoma cells . MATERIALS AND METHODS: Cell suspensions were transfected with different concentrations of the pEGFP-N1 plasmid . Shock-waves were applied in a degassed water bath with different numbers of impulses at different energy levels . Additionally, the effects of different DNA concentrations, frequencies and the absence/presence of a liquid air border were examined . RESULTS: After shock-wave application, the transfection rate increased up to a maximum of 27.10% after 1000 impulses at an energy level of 0.5 mJ/mm2 . In comparison negative control groups were transfected significantly below 1% . An increase in acoustic power and frequency and of DNA concentration and the presence of a liquid-air border resulted in an increasing transfection rate . CONCLUSION: The results demonstrate that naked plasmid DNA can easily and effectively be delivered to malignant urothelial cells in vitro upon exposure to lithotripter-generated shock-waves.

Mutagenesis, 2004 Mar, 19(2), 85 - 90
Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline Comet assay; Mukhopadhyay I et al.; The single cell gel electrophoresis (SCGE) assay, also known as the Comet assay, is one of the most promising genotoxicity tests developed in recent years to measure and analyse DNA damage in single cells . The present study was undertaken to assess the in vivo genotoxicity of the synthetic pyrethroid cypermethrin in brain ganglia and anterior mid gut of Drosophila melanogaster . Freshly emerged first instar larvae (22 +/- 2 h) were placed in different concentrations of cypermethrin (0.0004, 0.0008, 0.002, 0.2 and 0.5 p.p.m.) mixed in standard Drosophila food and allowed to grow . At 96 +/- 2 h, brain ganglia and anterior midgut from control and treated larvae were dissected out, single cell suspensions were prepared and a Comet assay was performed . Our results revealed a significant dose-dependent increase in DNA damage in the cells of brain ganglia and anterior midgut of D.melanogaster exposed to cypermethrin as compared with controls (P < 0.05 at 0.002 p.p.m.; P < 0.001 at 0.2 and 0.5 p.p.m.) . The present study shows in vivo genotoxicity of cypermethrin even at very low concentrations, which proves D.melanogaster as a model for in vivo genotoxicity assessment using the Comet assay.

Neuroscience, 2004, 124(3), 629 - 35
Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats; Ugrumov MV et al.; This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e . L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA . The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1% . The fetal substantia nigra containing DAergic neurons served as a control . DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine . Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses . L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra . Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus . This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons . Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.

Lab Anim, 2004 Jan, 38(1), 79 - 84
Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods; Chen JH et al.; Direct injection of VX2 cell suspension into the liver is simple and widely used . Implantation of a fragment of VX2 tumour into the liver using a surgical technique has also been developed in the last decade . In this study, we compared these two methods in order to find a better modality for establishing VX2 liver mass . Forty rabbits, each weighing 2.8-3.2 kg, were divided into two groups, 20 rabbits in each . In Group 1, a tumour cell suspension containing 1 x 10(6) cells in a volume of 0.1 ml, was injected slowly into the liver parenchyma using a 27-gauge needle during laparotomy . In Group 2, a 1 mm(3) fragment of VX2 carcinoma was inoculated into the sub-capsule of the left anterior lobe of the liver . In Group 1, three rabbits showed no tumour growth and 10 rabbits showed evidence of leakage and tumour seeding outside of the liver . In Group 2, all but one rabbit showed tumour growth and none showed evidence of tumour seeding . The leakage rates were 50% and 0% for Group 1 and Group 2, respectively . Overall, the success inoculation rate was 35% for Group 1 and 95% for Group 2 . In conclusion, to create the VX2 liver tumour model in rabbits, direct implantation of VX2 tumour fragment into the liver achieved better results than injecting cell suspension of VX2 tumour into the liver.

Methods Mol Biol, 2004, 263, 181 - 200
Hematopoietic stem cell characterization by Hoechst 33342 and rhodamine 123 staining; Bertoncello I et al.; A dual-dye efflux strategy utilizing the supravital dyes Hoechst 33342 (Ho) and rhodamine 123 (Rh123) is described and illustrated for the detection and analysis of hematopoietic stem cells in murine bone marrow . Mononuclear cells from bone marrow cell suspensions were incubated in a cocktail of Rh123 plus Ho, and both dyes were effluxed by two 15-min incubations in dye-free buffer prior to sorting . Compared to our original prototype method in which Rh123, but not Ho, was effluxed, this dual-dye efflux protocol more rapidly and efficiently resolves the most primitive Hodull/Rhdull hematopoietic stem cells . Moreover, under conditions of optimal dual-dye uptake and efflux, Hodull/Rhdull cells map to the subfraction of side population (SP) cells with the highest efflux of Ho, which were previously demonstrated to possess the highest hematopoietic stem cell activity.

Tree Physiol, 1986 Jun, 1(1), 21 - 30
Sustained division of protoplast-derived cells from primary leaves of Pinus pinaster, factors affecting growth and change in nuclear DNA content; David H et al.; Leaf protoplasts were isolated from apical and in vitro-induced axillary buds of Pinus pinaster Ait . seedlings . First divisions were seen after 8-10 days of culture in a 650 mOsm kg H(2)O(-1) medium in which glutamine was the sole nitrogen source . Colony formation was achieved in 6-7 weeks in a modified protoplast culture medium in which a reduction in the concentrations of both calcium and carbon was essential for sustained divisions . To maintain cell suspension growth, it was necessary to subculture every three weeks to a 170 mOsm kg H(2)O(-1) medium . Lowering the C/N ratio did not support better growth . Phenolic compounds were detected in stationary phase cultures . Analysis by HPLC indicated that the cinnamate pathway was involved in their synthesis . After 3 and 7 months of culture, 65 and 74%, respectively, of protoplast-derived cells had a nuclear DNA content comparable to that of leaf protoplasts.

Tree Physiol, 1989 Jun, 5(2), 259 - 66
Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus); Ochatt SJ et al.; Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues . Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15% . After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol . Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol . Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent . However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol . Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.

Tree Physiol, 1989 Dec, 5(4), 497 - 506
Growth and soluble proteins of cell cultures derived from explants and protoplasts of Pinus pinaster cotyledons; David H et al.; Pinus pinaster Ait . cell suspension cultures were derived from chopped cotyledons and from cotyledon protoplasts . When transferred after 12 weeks in culture, growth of both cell types showed a lag of 5 days followed by an exponential phase of 14 days for the protoplast-derived cells and 23 days for the organ-derived cells . During the exponential growth phase, packed cell volume of protoplast-derived cultures increased 7-fold and that of organ-derived cultures 13-fold . During the stationary phase, the diameters of protoplast-derived cells averaged 80 microm and those of organ-derived cells 100 microm . After 30 days, the media containing protoplast-derived and organ-derived cells decreased in osmolarity by 50 and 120 mOs per kg water, respectively, and in pH by 1.4 and 2.0 units, respectively . Throughout the growth cycle, protein content per unit of packed cell volume was always at least 35% higher in the protoplast-derived cultures than in the organ-derived cultures . Two-dimensional electrophoretic separation of soluble proteins revealed three peptides present in protoplast-derived cells that were absent from organ-derived cells and two peptides present in organ-derived cells that were absent from protoplast-derived cells . Other peptides differed quantitatively between the cell types.

Tree Physiol, 1988 Dec, 4(4), 371 - 80
The influence of glutamine on growth and viability of cell suspension cultures of Douglas-fir after exposure to polyethylene glycol; Leustek T et al.; The response of cell cultures of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) to osmotic stress was studied by measuring cell growth and viability after exposure to polyethylene glycol (PEG) (M(r) 6000-8000) . Growth of cells inoculated in a medium containing 10% PEG was slightly inhibited, whereas growth in a medium containing 15% PEG was severely inhibited . Cells grown for 6 days in nutrient medium and then subcultured in a medium containing 15% PEG to induce water stress showed high viabilities, whereas cells grown for longer than 6 days before exposure to PEG showed decreased viabilities after subculture . Cells grown in medium containing 30 mM glutamine were significantly more resistant to PEG-induced water stress, as measured by viability, than cells grown in medium without glutamine.

Tree Physiol, 1992 Apr, 10(3), 317 - 26
Effect of ABA on freezing resistance of Betula papyrifera and Alnus incana woody plant cell suspensions; Tremblay MF et al.; Treatment of birch (Betula papyrifera Marsh) and alder (Alnus incana (L.) Moench) cell suspension cultures with ABA increased the freezing resistance of the cells . After 7 days of treatment with 10(-5) M ABA, birch cells grown at 23 and 4 degrees C attained an LT(50) of -16.9 and -14.1 degrees C, respectively, whereas control cells had an LT(50) of -9.1 degrees C . In alder cell suspensions, treatment with 10(-5) M ABA at 23 degrees C induced a small increase in freezing resistance from -7.3 to -10.8 degrees C . Exposure to 4 degrees C alone did not induce a significant increase in hardiness in birch cell suspensions . Addition of 10(-5) M ABA to the medium inhibited fresh weight increase over 10 days of 3-g inocula of birch and alder by 70 and 52%, respectively . With the same concentration of ABA in the medium we found different intracellular ABA concentrations in 3- and 6-g inocula . We conclude that the concentration of ABA in the medium does not reflect the intracellular concentration of tissue cultures, and that cultural conditions may influence ABA accumulation by cell cultures.

Life Sci, 2004 Mar 12, 74(17), 2111 - 28
Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells; Gooz M et al.; This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms . OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay . Nicotine (10(-6) - 10(-3) M) stimulated significant OLF secretion in rat adrenocortical cells . Acetylcholine (10(-7) - 10(-4) M) and eserine (10(-7) - 10(-3) M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations . Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion . ACTH (10(-8) M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent . The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion . The alpha7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion . These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.

Cryobiology, 2004 Feb, 48(1), 8 - 21
Direct cell injury associated with eutectic crystallization during freezing; Han B et al.; Freezing induced direct cell injury has been explained by a two-factor hypothesis-intracellular ice formation (IIF) at rapid cooling rates, and solution effects at slow cooling rates . Even though IIF is generally believed to be a major injury mechanism at rapid cooling rates, injury by solution effects is not fully understood and several injury mechanisms have been suggested . Solution effects have generally been considered the result of the elevated electrolyte concentration within the intracellular and extracellular space during freezing . In addition to the injury by this elevated electrolyte concentration, freezing injury associated with eutectic crystallization was investigated . To examine the injury associated with eutectic crystallization, two different experiments were designed and performed . In the first experiment, two groups of AT-1 rat prostate tumor cell suspensions were frozen and thawed on a cryomicroscope in the same way except that eutectic crystallization was initiated in only one group . During the second experiment, AT-1 cells were suspended in several different media, which have different eutectic crystallization temperatures, and exposed to a single cooling-warming cycle with varying end temperature of the protocol on a directional solidification stage . After both experiments, post-thaw viability was evaluated and compared . The post-thaw viability drops significantly upon the occurrence of the eutectic crystallization regardless of suspending media, which suggests direct cell injury associated with eutectic crystallization . Based on these observations, two possible injury mechanisms are anticipated: (i) mechanical damage to the cell membrane due to eutectic crystallization, and (ii) intracellular eutectic formation (IEF) . The proposed mechanisms provide a more comprehensive physical explanation of freezing induced cell injury and extend the understanding on solution effects.

J Agric Food Chem, 2004 Feb 25, 52(4), 972 - 9
Specific synthesis of 5,5'-dicapsaicin by cell suspension cultures of capsicum annuum var . annuum (chili Jalapeño chigol) and their soluble and NaCl-extracted cell wall protein fractions; Martinez-Juarez VM et al.; HPLC-UV, (1)H NMR, (13)C NMR, and (1)H-(1)H COSY analyses revealed that exogenous capsaicin was specifically converted into 5,5'-dicapsaicin by both cell suspension cultures of Capsicum annuum var . annuum (chili Jalapeno chigol) and their soluble and NaCl-extracted cell wall protein fractions under oxidative conditions . In cell suspension cultures 5,5'-dicapsaicin was found only in biomass of capsaicin-fed cultures . This compound has not been detected before either in fresh fruits or in in vitro cultures of Capsicum . The transformation of capsaicin by different protein fractions revealed that most of the enzymatic activity was located in the NaCl-extracted, or ionic cell wall bound, protein, and that it was strictly dependent on H(2)O(2) . These results might in part explain some previously described features of capsaicin production by in vitro cultures of Capsicum . The implications of the results regarding the catabolism of capsaicinoids are discussed.

Biorheology, 2004, 41(1), 29 - 43
Conductometric study of shear-dependent processes in red cell suspensions . II . Transient cross-stream hematocrit distribution; Pribush A et al.; A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel . The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance . Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level . The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate . A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening . It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks . For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.

Biorheology, 2004, 41(1), 13 - 28
Conductometric study of shear-dependent processes in red cell suspensions . I . Effect of red blood cell aggregate morphology on blood conductance; Pribush A et al.; The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz . The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks . It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates . Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis . These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Feb, 24(2), 192 - 4, 197
In vitro culture and induced differentiation of adult rat neural stem cells from the corpus striatum; Wang YH et al.; OBJECTIVE: To investigate the in vitro multipotential differentiation of neural stem cells from adult rat corpus striatum . METHODS: The neural stem cells isolated from adult rat corpus striatum were cultured in serum-free medium to obtain cell suspension before monoclonal subculturing and differential induction . Immunocytochemical staining and reverse transcriptional PCR (RT-PCR) were performed to identify the properties of the differentiated cells . RESULTS: Numerous cell clusters were formed in the phase of monoclonal culture, and different types of cells were observed 3 d after induction with fetal bovine serum . The differentiated cells contained cells positive for nestin, neuron-specific enolase (NSE) positive cells, and glial fibrillary acidic protein (GFAP) positive cells . RT-PCR identified expressions of the transcripts for neural cell-associated genes including brain factor-1, gamma-aminobutyric acid alpha-receptor gamma-subunit, tyrosine hydroxylase and tryptophan hydroxylase . CONCLUSION: The cells separated from adult rat corpus striatum possess the ability of self-proliferation and multipotential differentiation, and are identified as the stem cells of the central nervous system.

Genet Mol Res, 2002 Jun 30, 1(2), 117 - 27
Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis . Application in tissue sections and cell suspensions; Muhlmann M; As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase . This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe) . The probe, bound to the target, will be developed into a fluorescent signal . The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples . FISH is still used mostly in research, but there are diagnostic applications . New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH . Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases . In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes . CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients.

Acta Neuropathol (Berl), 2004 May, 107(5), 421 - 7 Epub 2004 Feb 11.
Fetal allogeneic dopaminergic cell suspension grafts in the ventricular system of the rat: characterization of transplant morphology and graft-host interactions; Oertel J et al.; Experimental transplantation trials of fetal cells in Parkinson's and Huntington's disease or multiple sclerosis still require allogeneic graft material and raise questions of graft rejection and immunosuppression . Alternatively to the striatum, the lateral ventricles have been discussed as grafting site in Parkinson's and Huntington's disease although little is known of the specific immunology of the ventricular system . To address this question, 28 adult female LEW1.W rats received intraventricular allogeneic dopaminergic cell suspension grafts from E14 DA rat fetuses . Twelve animals with syngeneic grafts served as control . Immunohistochemical examination was performed with staining for MHC expression, microglia-macrophages, various lymphocyte subsets, dopaminergic neurons and astrocytes at 4 days, and 1, 3, 6, and 12 weeks after transplantation . In all animals, intraventricular transplants were found, which showed maturation and integration in the host parenchyma at the later time points . Animals with allogeneic grafts developed a vivid immune response with strong MHC class I expression and dense lymphocyte infiltrates . Surprisingly, this immune response subsided at 12 weeks and healthy grafts remained . These results indicate (1) that, in contrast to intraparenchymal grafts, a strong immune response to allogeneic fetal cell suspension grafts can be elicited by intraventricular grafting, (2) that a peculiar immunological role of the ventricular system has to be considered in further studies, and (3) that a vivid immune response to allografts in the brain may subside without graft destruction.

Planta, 2004 May, 219(1), 73 - 83 Epub 2004 Feb 11.
Extracellular cross-linking of xylan and xyloglucan in maize cell-suspension cultures: the role of oxidative phenolic coupling; Kerr EM et al.; Cell-suspension cultures of maize ( Zea mays L.) released soluble extracellular polysaccharides (SEPs) into their medium . Some or all of the SEPs had feruloyl ester groups . Pulse-labelling with {(3)H}arabinose was used to monitor changes in the SEPs' M(r) (estimated by gel-permeation chromatography) with time after synthesis . Newly released (3)H-SEPs were 1.3-1.6 MDa, but between 2 days and 3 days after radiolabelling (in one experiment) or between 5 days and 6 days (in another), the (3)H-SEPs abruptly increased to approximately 17 MDa, indicating extensive cross-linking . The cross-linking involved both {(3)H}xylan and {(3)H}xyloglucan components of the SEPs . The cross-links could be cleaved by alkali, returning the SEPs to their original M(r) . In 0.1 M NaOH at 37 degrees C, 58% cleavage was effected within 24 h . The requirement for such prolonged alkali treatment indicates that ester-bonded (e.g . diferuloyl) groups were not solely responsible for the cross-linking . Bonds cleaved only by relatively severe alkali could include benzyl ether linkages formed between sugar residues and oxidised phenolics that had quinone methide structures . The ability of alkali to cleave the cross-links was independent of the age of the (3)H-SEP molecules . Cross-linking of (3)H-SEPs in vivo was delayed (up to approx . 7 days after radiolabelling) by exogenous sinapic acid, chlorogenic acid or rutin-agents predicted to compete with the oxidative coupling of feruloyl-polysaccharides . The cross-linking was promoted by exogenous ferulic acid or l-tyrosine, possibly because these compounds acted as precursors for polysaccharide feruloylation, thus providing additional partner substrates for the oxidative coupling of previously formed (3)H-SEPs . The ability of certain phenolics to prevent the cross-linking of (3)H-SEPs supports the idea that the cross-linking involved phenolic oxidation.

Photochem Photobiol Sci, 2004 Feb, 3(2), 211 - 6 Epub 2003 Nov 12.
Photobiological modulation of cell attachment via cytochrome c oxidase; Karu TI et al.; The number of cells attached to glass substrates increases if HeLa cell suspensions are irradiated with monochromatic visible-to-near infrared radiation (600-860 nm, 52 J m(-2)) prior to plating . The well-structured relationship between this biological response and the radiation wavelength (action spectrum with maxima at 620, 680, 760, and 820 nm) suggests the existence of a photoacceptor responsible for the enhancement of attachment (presumably cytochrome c oxidase, the terminal enzyme of the respiratory chain) and, secondly, the existence of signaling pathways between the mitochondria, the plasma membrane, and the nucleus of the cell . Treating the cell suspension with ouabain (a Na(+), K(+)-ATPase inhibitor), amiloride (an inhibitor of N(+)/H(+) exchangers), or sodium azide (a cytochrome c oxidase inhibitor) prior to irradiation significantly modifies the action spectrum of cell attachment enhancement . The action of the chemicals under study also depends on their concentration and radiation fluence . Our results point to the existence of at least three signaling pathways (reaction channels) relating together the cell attachment, the respiratory chain, and the Na(+), K(+)-ATPase and N(+)/H(+) exchanger activities.

Eur J Oral Sci, 2004 Feb, 112(1), 48 - 54
T-cell costimulatory capacity of oral and skin epithelial cells in vitro: presence of suppressive activity in supernatants from skin epithelial cell cultures; Hasseus B et al.; Oral Langerhans cells (LC) have better T-cell costimulatory capacity than skin LC . In this study factors affecting this capacity have been assessed in a mixed epithelial cell lymphocyte reaction (MELR) assay . Flow cytometry analysis of freshly recovered cells revealed major histocompatibility complex (MHC) class II molecule expression on 7.5% of the oral epithelial cells and 9.7% of the skin epithelial cells . Monoclonal anti class II antibodies significantly reduced the T-cell proliferation in the MELR . Pretreatment of skin epithelial cells with interleukin-1beta, tumour necrosis factor-alpha or interferon (IFN)-gamma did not affect the MELR proliferation, but incubation with IFNgamma significantly suppressed the T-cell response . Transfer of supernatants from cultures of skin epithelial cells and allogeneic T cells to cultures of oral epithelial cells and T cells resulted in a reduced T-cell proliferation while supernatants from oral epithelial cells and T cells did not reduce proliferation . The higher proliferation in cultures of T cells and oral epithelial cells than in cultures containing skin epithelial cells may be due to the presence of a suppressive factor in the skin epithelial cell suspensions.

Exp Neurol, 2003 Dec, 184(2), 615 - 35
Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions; Eriksson C et al.; In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes . In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons . Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse . Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats . Cell suspensions of primary LGE tissue were prepared and grafted in parallel . Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes . Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons . Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb . MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus . The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.

Biol Reprod, 2004 Jun, 70(6), 1738 - 50 Epub 2004 Feb 06.
Clonogenicity of human endometrial epithelial and stromal cells; Chan RW et al.; The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer . Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property . The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones . Endometrial tissue was obtained from women undergoing hysterectomy . Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors . Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium . The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells . In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect . TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect . Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin . All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified . This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.

Cell Transplant, 2003, 12(8), 891 - 6
Hepatic cells via cava vein can influence allogenic islet rat transplantation; Jara-Albarran A et al.; We have reported, previously, some effect of allogenic hepatic cells for islet tolerance when they are injected mixed (hepatic cells and islets) in different proportions via portal vein, in diabetic Wistar rats . Now we have studied the role of allogenic hepatic cells injected sequentially 15 min before islets, comparing via the portal vein (A and B groups) and via the cava vein (C and D groups) with a control group of islets alone . The allogenic islets were always injected via portal vein, in similar conditions, while the ratio of hepatic cells/islets was 100:1 (A, C groups) or 200:1 (B, D groups) . Islets and hepatic cells were obtained from several different rats . The transplanted rats were observed during 30 days and results compared among the different rat groups: porta-porta (P/P), cava-porta (C/P), and control group . Statistically, a significant interaction between type of transplant and proportion of hepatic cells was observed . Also, C plus D groups showed statistical difference with the control group (p < 0.017) and also all the groups together (p < 0.047) . These results suggest that hepatic cells can induce, in some cases, islet graft prolongation in Wistar rats . Better results were obtained when hepatic cells were injected via the cava vein than via the portal vein . Because we used a liver cell suspension integrated for several kinds of cells, the study does not clarify if this effect can be related to some specific hepatic cell subpopulation . To confirm the results and to determine if the hypothetical mechanism can be attributed to a block of the immune system or to some factor secreted by hepatic cells, more studies must be performed.

Ann Surg Oncol, 2004 Feb, 11(2), 147 - 56
Intratumoral IL-12 and TNF-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity; Sabel MS et al.; BACKGROUND: Local, sustained delivery of cytokines at a tumor can enhance induction of antitumor immunity and may be a feasible neoadjuvant immunotherapy for breast cancer . We evaluated the ability of intratumoral poly-lactic-acid-encapsulated microspheres (PLAM) containing interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) in a murine model of breast cancer to generate a specific antitumor response . METHODS: BALB/c mice with established MT-901 tumors underwent resection or treatment with a single intratumoral injection of PLAM containing IL-12, TNF-alpha, or GM-CSF, alone or in combination . Two weeks later, lymph nodes and spleens were harvested, activated with anti-CD3 monoclonal antibodies (mAb) and rhIL-2, and assessed for antitumor reactivity by an interferon gamma (IFNgamma) release assay . Tumor-infiltrating lymphocyte (TIL) analysis was performed on days 2 and 5 after treatment by mechanically processing the tumors to create a single cell suspension, followed by three-color fluorescence-activated cell sorter (FACS) analysis . RESULTS: Intratumoral injection of cytokine-loaded PLAM significantly suppressed tumor growth, with the combination of IL-12 and TNF-alpha leading to increased infiltration by polymorphonuclear cells and CD8+ T-cells in comparison with controls . The induction of tumor-specific reactive T-cells in the nodes and spleens, as measured by IFN-gamma production, was highest with IL-12 and TNF-alpha . This treatment resulted in resistance to tumor rechallenge . CONCLUSIONS: A single intratumoral injection of IL-12 and TNF-alpha-loaded PLAM into a breast tumor leads to infiltration by polymorphonuclear cells and CD8+ T-cells with subsequent tumor regression . In addition, this local therapy induces specific antitumor T-cells in the lymph nodes and spleens, resulting in memory immune response.

J Endod, 2004 Jan, 30(1), 25 - 9
Attachment and morphological behavior of human periodontal ligament fibroblasts to mineral trioxide aggregate: a scanning electron microscope study; Balto HA; The attachment and morphology of human periodontal ligament fibroblasts to mineral trioxide aggregate (MTA) was evaluated using a scanning electron microscope . The material was placed at an apical cavity of 30 single-rooted slices of extracted human teeth . The specimens were divided into two groups of 15 root slices each (freshly mixed and set state) . For each experimental group, five root slices were used per observation period (4, 8, and 24 h) . A set of two glass slides was used per observation period for the control group . The experiments were performed in tissue-culture cluster 96-well plates in which 1 ml of human periodontal ligament fibroblast cell suspension was placed over the MTA filling and the control glass slides . For the positive-control group, 0.5 ml of methyl methacrylate 2% (vol/vol) was added to the cell suspensions before being dispensed into the wells . Results showed the normal cell morphology in the negative controls . Few round cells with less smooth surfaces and many rough blebs were seen in the positive control, and most of these cells did not show any attachment to the substratum . Similar observations were seen with the freshly prepared-MTA group . In the set-MTA group, cells were round and flattened, displayed smooth surfaces, and appeared to be tightly attached to MTA . It was concluded that the quality and quantity of cell attachment to the retrofilling material could be used as a criterion to evaluate material's toxicity . This research (FN#1077) is registered with the College of Dentistry research center, King Saud University, Riyadh, Saudi Arabia . The author thanks the administration of the King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, and in particular Dr . M . N . Al-Ahdal for providing the use of the Molecular Virology and Infectious Disease Laboratory, Mr . Yunus Siddiqui for his support, and Dr . Saad AL-Nazhan for his assistance in preparing the manuscript.

World J Gastroenterol, 2004 Feb 1, 10(3), 433 - 6
Extraction of protoporphyrin disodium and its inhibitory effects on HBV-DNA; Li CP et al.; AIM: To explore an ideal method for extracting protoporphyrin disodium (PPN) from unanticoagulated animal blood, and to study the inhibitory effects of PPN on HBV-DNA duplication and its cytotoxicity to 2.2.15 cell strain . METHODS: Protoporphyrin methyl ester and other intermediate products were prepared with protoheme separated from protein hydrolysates of coagulated animal blood, which were finally made into PPN and detected quantitatively with an ultraviolet fluorescent analyzer . Ten microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml of PPN-aqueous solution were added into culture medium for 2.2.15 cells respectively . Eight days later, the drug concentration in supernatant from the culture medium was detected when inhibition rate of HBeAg, cell survival rate when inhibition rate of HBeAg was 50% (ID50), and when survival cells in experimental group were 50% of those in control group (CD50), and the therapeutic index (TI) was also detected . PPN with different concentration of 10 microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml was respectively mixed and cultivated with HepG2 2.2.15 cell suspension, and then the inhibition of PPN against HBV-DNA was judged by PCR . RESULTS: The extract of henna crystal was identified to be PPN . When the concentrations of PPN were 160 microg/ml and 80 microg/ml, the inhibition rates of HBeAg were 89.8% and 82.4%, and the cell survival rates were 98.7% and 99.2% . CONCLUSION: It is suggested that PPN can be extracted from unanticoagulated animal blood . PPN can inhibit HBV-DNA expression and duplication in vitro, and has no cytotoxicity to liver cells . Further study and application of PPN are warranted.

Space Med Med Eng (Beijing), 2003 Oct, 16(5), 332 - 5
{Circadian rhythms of DNA synthesis and telomerase expression in hepatic cancer transplanted in nude mice}; Qu Y et al.; Objective: To study the circadian rhythms of DNA synthesis and telomerase expression in hepatic cancer transplanted in nude mice . Method: Sixteen BALB/C mice were synchronized with an alternative lighting regimen with 12 h for light and 12 h for darkness (12:12 LD) for 4 weeks . Hepatic cancer cells (SMMC-7721) were implanted into both flanks of each mouse . One week after transplantation, sampling from the tumor was conducted at 3, 9, 15 and 21 h after light onset (HALO) . Single cell suspension was obtained and stained with propidium iodide . The cellular DNA content was measured by flow cytometry . Telomerase activity was measured by PCR-ELISA assay . Data were documented by ANOVA and Cosinor analysis . Result: The proportion of tumor cells in phase G1, S, G2/M and telomerase activity varied according to circadian time with statistical significance, and the telomerase activity showed a synchronized variation . The distribution curves of both phase S and the expression level of telomerase were fit for Cosinor changes . Conclusion: DNA synthesis and telomerase expression of SMMC-7721 cells transplanted into the nude mice varies according to the circadian rhythm . The results provide a guidance for laying down the chemotherapy protocol for human tumors, especially when the telomerase inhibitor was used as the anti-cancer agent.

Biochem J, 2004 May 1, 379(Pt 3), 601 - 7
Identification and characterization of plant glycerophosphodiester phosphodiesterase; Van Der Rest B et al.; GPX-PDE (glycerophosphodiester phosphodiesterase; EC 3.1.4.46) is a relatively poorly characterized enzyme that catalyses the hydrolysis of various glycerophosphodiesters (glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoserine and bis-glycerophosphoglycerol), releasing sn-glycerol 3-phosphate and the corresponding alcohol . In a previous study, we demonstrated the existence of a novel GPX-PDE in the cell walls and vacuoles of plant cells . Since no GPX-PDE had been identified in any plant organism, the purification of GPX-PDE from carrot cell walls was attempted . After extraction of cell wall proteins from carrot cell suspension cultures with CaCl2, GPX-PDE was purified up to 2700-fold using, successively, ammonium sulphate precipitation, gel filtration and concanavalin A-Sepharose . Internal sequence analysis of a 55 kDa protein identified in the extract following 2700-fold purification revealed strong similarity to the primary sequence of GLPQ, a bacterial GPX-PDE . To confirm the identity of plant GPX-PDE, an Arabidopsis thaliana cDNA similar to that encoding the bacterial GPX-PDE was cloned and overexpressed in a bacterial expression system, and was used to raise antibodies against the putative Arabidopsis thaliana GPX-PDE . Immunochemical assays performed on carrot cell wall proteins extracted by CaCl2 treatment showed a strong correlation between GPX-PDE activity and detection of the 55 kDa protein, validating the identity of the plant GPX-PDE . Finally, various properties of the purified enzyme were investigated . GPX-PDE is a multimeric enzyme, specific for glycerophosphodiesters, exhibiting a K(m) of 36 microM for glycerophosphocholine and active within a wide pH range (from 4 to 10) . Since these properties are similar to those of GLPQ, the bacterial GPX-PDE, the similarities between plant and bacterial enzymes are also discussed.

Technol Cancer Res Treat, 2004 Feb, 3(1), 1 - 14
Polarized reflectance spectroscopy for pre-cancer detection; Sokolov K et al.; Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life . Thus, highly selective, sensitive and cost-effective screening and diagnostic techniques to identify curable pre-cancerous lesions are desperately needed . Precancers are characterized by increased nuclear size, increased nuclear/cytoplasmic ratio, hyperchromasia and pleomorphism, which currently can only be assessed through an invasive, painful biopsy . Here, we describe the development of a non-invasive optical technique based on polarized reflectance spectroscopy that has the potential to provide in real time diagnostically useful information for pre-cancer detection . Our results demonstrate that polarized reflectance spectroscopy can be used to selectively detect the size-dependent scattering characteristics of nuclei in vivo . We gradually progress from cell suspensions to realistic three-dimensional tissue models of epithelium, then to cervical biopsies and, finally to in vivo studies on normal volunteers and clinical patients.

Biol Blood Marrow Transplant, 2004 Feb, 10(2), 135 - 41
The role of depletion of dimethyl sulfoxide before autografting: on hematologic recovery, side effects, and toxicity; Syme R et al.; Cryopreservation of stem cells after collection from peripheral blood or bone marrow for autologous transplantation necessitates protection with dimethyl sulfoxide (DMSO) . Unfortunately, DMSO, when infused with the thawed cell suspension, may induce serious complications and side effects . To assess whether depletion of DMSO before autografting affects safety and efficacy, 56 consenting consecutive patients treated with high-dose chemotherapy and autologous blood stem cell transplantation were assigned to obtain either an untreated or DMSO-depleted autograft . On the day of transplantation, the cryopreserved cells were thawed and infused to the patient either immediately or after washing 3 times in normal saline supplemented with 6% anticoagulant citrate dextrose solution . Cell count with viability, clonogenic assay, and phenotyping were performed before and after thawing and after washing . Hematologic recovery, side effects, and complications were recorded . The in vitro and clinical data on 56 patients show that the depletion of DMSO in vitro before autografting does not induce a significant loss of cell number, viability, colony-forming unit-granulocyte-macrophage activity, or number of CD34(+) cells . Furthermore, it leads to a safe and sustained engraftment . The complications and side effects, as recorded by continuous monitoring, were substantially less; however, the procedure takes 3 to 4 hours of laboratory work per patient.

Obes Res, 2004 Jan, 12(1), 95 - 105
Computerized determination of adipocyte size; Bjornheden T et al.; OBJECTIVE: Fat cell size is a fundamental parameter in the study of adipose tissue metabolism, because it markedly influences the cellular rates of metabolism . Previous techniques for the sizing of adipocytes are often complicated or time-consuming . The aim of this study was to develop a new, computerized method for rapid and accurate determination of adipocyte size in a cell suspension obtained by incubating human or rat adipose tissue biopsies with collagenase . RESEARCH METHODS AND PROCEDURES: The cell suspension was placed between a siliconized glass slide and a cover slip . Using the reference method {designated as (R)}, the cell diameters were determined manually using a microscope with a calibrated ocular . The new method presented here {designated as (C)} was based on computerized image analysis . RESULTS: After two well-defined corrective adjustments, measurements with (R) and (C) agreed very well . The small remaining differences seemed, in fact, to depend on inconsistencies in (R) . DISCUSSION: We propose that (C) constitutes a valuable tool to study fat cell size, because this method is fast and allows the assessment of a sufficient number of cells to get reliable data on size distribution . Furthermore, images of cell preparations may be stored for future reference.

Biogerontology, 2003, 4(6), 365 - 70
The effect of carbon tetrachloride and ultraviolet radiation on dolichol levels in liver cells isolated from 3- and 24-month-old male Sprague-Dawley rats; Parentini I et al.; Dolichol (D) is a long-chain polyprenoid broadly distributed in the cell membranes, possibly endowed with a free-radical scavenging activity, whose concentration in tissues increases with increasing age . No enzyme pathway for D degradation has been discovered . In order to test the hypothesis that D might undergo a non-enzymatic free-radical mediated decomposition the effects of a xenobiotic agent (carbon tetrachloride, CCl(4)) and ultraviolet-B (UV-B) radiation on D levels were studied in liver cells isolated from male ad libitum fed Sprague-Dawley rats aged 3 or 24 months . Liver cells (90 mg/ml) were incubated in sealed flasks (6 ml cell suspension each) for 0, 5, 10 and 20 min after the addition of 25, 50 or 200 microl CCl(4) in the central well . 50 ml of a 6 mg/ml liver cell suspension were poured in a 120 cm(2) Petri dish and the sediment liver cell monolayer was exposed to UVB radiation for 0, 5, 10, 20 and 40 min . At the given time, cells were taken and D was extracted and assayed by the HPLC procedure . D levels were remarkably higher in older than in younger cells as expected ( P < 0.001) . Treatment with CCl(4) and UVB caused a highly significant decrease in D ( P < 0.001) whose percentage was larger in younger than in older cells . The conclusions are that free-radicals generated either by chemical or by physical agents cause a very rapid depletion of D in liver cells, and that the effect of the free radical attack on D decomposition may be lower percentage wise in older than in younger cells, which might account at least in part for the accumulation of D in older tissues.

Science, 2004 Feb 27, 303(5662), 1352 - 5 Epub 2004 Jan 22.
Selective differentiation of neural progenitor cells by high-epitope density nanofibers; Silva GA et al.; Neural progenitor cells were encapsulated in vitro within a three-dimensional network of nanofibers formed by self-assembly of peptide amphiphile molecules . The self-assembly is triggered by mixing cell suspensions in media with dilute aqueous solutions of the molecules, and cells survive the growth of the nanofibers around them . These nanofibers were designed to present to cells the neurite-promoting laminin epitope IKVAV at nearly van der Waals density . Relative to laminin or soluble peptide, the artificial nanofiber scaffold induced very rapid differentiation of cells into neurons, while discouraging the development of astrocytes . This rapid selective differentiation is linked to the amplification of bioactive epitope presentation to cells by the nanofibers.

Biol Reprod, 2004 May, 70(5), 1428 - 37 Epub 2004 Jan 21.
Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas; He Y et al.; In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas . This represents the first application of the DSC technique to sperm cells from nonmammalian species . Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA) . Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide . Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide . Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume . By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined . The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole) . The corresponding parameters in the presence of 8% DMSO were: Lpg{cpa} = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp{cpa} = 38.0 kJ/mole (9.1 kcal/mole) . Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole) . The corresponding parameters in the presence of 8% DMSO were: Lpg{cpa} = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp{cpa} = 37.6 kJ/mole (9.0 kcal/mole) . The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min . These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C . gigas.

Pest Manag Sci, 2004 Jan, 60(1), 65 - 74
Characterization of the bound residues of the fungicide cyprodinil formed in plant cell suspension cultures of wheat; Sapp M et al.; The non-extractable residues of the fungicide cyprodinil formed in heterotrophic cell suspension cultures of wheat were studied by application of {2-pyrimidyl-14C} or {2-pyrimidyl-13C}cyprodinil . The main objective was to examine whether solid-state and liquid 13C NMR spectroscopy can be used to examine plant bound residues of pesticides . For 14C experiments, wheat suspensions grown on glucose as carbon source were treated with 10 mg litre(-1) of 14C-cyprodinil . After incubation for 12 days, 20% of applied 14C was detected as non-extractable residues . The cell debris were treated with 0.1 M HCl (reflux), 1.0 M HCl (reflux), buffer, or 2 M NaOH (50 degrees C); Bjorkman lignin and acidolysis lignin fractions were also prepared from the debris . Radioactivity liberated and solubilized by these procedures was examined by thin-layer chromatography and high-performance liquid chromatography . The results showed that cyprodinil and primary metabolites contributed to the fungicide's bound residues . Most of the residues (12% of applied 14C) remained associated with polar or polymeric/oligomeric endogenous cell materials in a stable manner . For the study with 13C-cyprodinil, wheat suspensions were cultivated on 13C-depleted glucose for four growth cycles, resulting in maximum 13C depletion of the natural cell components to about 0.10% . During the fourth cycle, 13C-labelled cyprodinil was applied, and cells were incubated (12 days) . Cell debris was prepared and examined by solid-state 13C NMR spectroscopy . Debris was then treated as described above in the 14C experiment . Solubilized fractions were analyzed by liquid 13C NMR spectroscopy . However, none of the 13C NMR spectra recorded gave utilizable or unambiguous results, and all exhibited large inconsistencies, especially concerning the data from the conventional 14C experiment.

J Biol Chem, 2004 Mar 26, 279(13), 13044 - 53 Epub 2004 Jan 13.
Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators; Hanson GT et al.; Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue . This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences . By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created . roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo . Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes . roGFP1 has been targeted to the mitochondria in HeLa cells . Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants . The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH approximately 8.0 at 37 degrees C) . In other work (C . T . Dooley, T . M . Dore, G . Hanson, W . C . Jackson, S . J . Remington, and R . Y . Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

Zhonghua Yi Xue Za Zhi, 2003 Dec 25, 83(24), 2162 - 5
{Endobronchial eosinophils preferentially stimulate T helper cell type 2 responses}; Shi HZ et al.; OBJECTIVE: The aim of the present study was to evaluate whether eosinophils within the tracheobronchial lumen can stimulate Th2 cell expansion by presenting antigen both in vitro and in vivo . METHODS: Airway eosinophils were recovered from ovalbumin-sensitized and -challenged BALB/c mice, these eosinophils were then cocultured with sensitized CD4(+) cells in the absence or presence of anti-CD80 or/and -CD86 monoclonal antibodies . Airway eosinophils were instilled into the trachea of sensitized mice, At 3 d thereafter, the draining paratracheal lymph nodes were removed and teased into cell suspensions for culture . Cell-free culture supernatants were collected for detection of cytokines . RESULTS: Our data showed that airway eosinophils, recovered following inhalational ovalbumin challenge in sensitized mice functioned as CD80- and CD86-dependent antigen-presenting cells to stimulate sensitized CD4(+) lymphocytes to produce IL-4, IL-5 and IL-13, but not interferon-gamma (IFN-gamma) in vitro assay . When instilled intratracheally in ovalbumin-sensitized recipient mice, these antigen-loaded eosinophils migrated into draining paratracheal lymph nodes primed Th2 cells in vivo for IL-4, IL-5 and IL-13, but not IFN-gamma, production during the in vitro culture that was also CD80- and CD86-dependent . CONCLUSIONS: We concluded that eosinophils within the lumina of airways could process inhaled antigen function in vitro and in vivo as antigen-presenting cells to promote expansion of Th2 cells . This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate immune responses by amplifying Th2 cell responses.

J Neurochem, 2004 Feb, 88(3), 698 - 707
Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347; Boll JB et al.; Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD) . A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients . Studies in rats indicated that many grafted neurones die by apoptosis . CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis . We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats . CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress . Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats . Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth . Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.

Biotechnol Lett, 2003 Dec, 25(23), 2023 - 8
Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa; Jeffers P et al.; Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa . Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles . For the heavily aggregated C . calcitrapa, biomass levels {from 10-160 g fresh weight (fw) l(-1))} were linearly correlated with FBRM counts . For M . citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length . For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis . The average aggregate equivalent diameter (C . calcitrapa) and average chain length (M . citrifolia), determined using image analysis, closely tracked the FBRM average chord length . The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.

J Microencapsul, 2004 Feb, 21(1), 15 - 24
Survival of Beijerinckia sp . microencapsulated in carbohydrates by spray-drying; Boza Y et al.; The encapsulation of Beijerinckia sp . cell suspension in different wall materials using the spray drying technique was performed . Mat dextrin, dehydrated glucose syrups, gum acacia and modified starch materials were tested . Cell viability assays were carried out before and after drying and during storage of the products . The surface area and characteristics of the encapsulated powders were examined using BET adsorption of N(2) and scanning electron microscopy, respectively . The residual moisture content and water activity of the powders were also determined . The best results were obtained with the dehydrated glucose syrup, which resulted in products with the greatest per cent survival during the drying process and subsequent storage period . The products obtained with the dehydrated glucose syrup showed more uniform microcapsule surfaces at lower A(w) values and residual moisture content.

Pigment Cell Res, 2004 Feb, 17(1), 62 - 5
Morphology of cultured human epidermal melanocytes observed by atomic force microscopy; Zhang RZ et al.; The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM) . Epidermis obtained from human foreskins was treated with 0.5% dispase . Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed . Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature . Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose . These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions . We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed . In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes . The termini of dendrites formed unbranched terminal protrusions (approximately 1,500-2,000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards . The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes . We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts . The melanin granules were expelled by exocytosis.

Pigment Cell Res, 2004 Feb, 17(1), 51 - 61
Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice; Hirobe T et al.; Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF) . Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion) . The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes . Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF . Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes . These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.

Biotech Histochem, 2003 Jun-Aug, 78(3-4), 171 - 7
Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions; Wagner SJ et al.; Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur . One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation . The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components . Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark . Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue . Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage . In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing . Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue . Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased . Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution . Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage . Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.

Urol Res, 2004 Aug, 32(4), 255 - 60 Epub 2004 Jan 09.
Micelle delivery of doxorubicin increases cytotoxicity to prostate carcinoma cells; McNealy TL et al.; The use of doxorubicin as a chemotherapeutic agent is hindered by its toxic side effects on the normal cells of the body . The objective of this study was to determine if micelle-delivered doxorubicin could increase the effectiveness of doxorubicin against prostate carcinoma cells . Rat prostate carcinoma cells (MatLu) were cultured under standard conditions . Phosphate-buffered saline (PBS), doxorubicin and/or micelle solution (Pluronic 10500 solution) was added to the cell suspensions and incubated for 3 h . After incubation, cells were washed twice . Analysis consisted of: 1) immediate cell count and 2) proliferation assay at 24 and 144 h . After 24 h, samples with micelle-incorporated doxorubicin had 75% (10% pluronic with 10 microg/ml doxorubicin) and 80% (1% pluronic with 10 microg/ml doxorubicin) cell proliferation results compared with the control group . After 144-h incubation, these same two groups demonstrated cell proliferation results of only 30 and 43% of the control group . The in vitro cytotoxicity of doxorubicin against prostate carcinoma cells was dramatically increased by incorporating the molecule with polymeric micelles .

J Urol, 2004 Feb, 171(2 Pt 1), 944 - 9
Urogenital tract expression of enhanced green fluorescent protein in transgenic mice driven by a smooth muscle gamma-actin promoter; Szucsik JC et al.; PURPOSE: Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation . We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays . MATERIALS AND METHODS: A 13.7 kb mouse smooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei . Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies . In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting . RESULTS: Six lines of transgenic mice were generated (transgene copy numbers 1 to 30) . EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels . EGFP levels appeared to correlate with transgene copy number . Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined . Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis . CONCLUSIONS: Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained . The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes . These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.

Klin Lab Diagn, 2003 Nov, (11), 35 - 9
{Immunophenotyping in diagnosis of chronic lymphoproliferative diseases}; Samoilova RS et al.; With due respect to their many-year experience, the authors focused their attention on the peculiarities of implementation and registration aspects of the immunofluorescence method of immunotyping made in cell suspensions and in tissue imprints; additionally they made a system of a set of monoclonal antibodies (used for the purpose), which enable the differential diagnosis of reactive conditions related with various malignant lymphoproliferative diseases (LPD) . It order to specify a nature of lymphoid cells it is suggested to undertake the immunophenotyping withing a gradually expanding set of monoclonal antibodies, which reflects different parameters of lymphoid cells like linear attributes, clonal characteristics, differential-diagnostic markers, functional status and proliferative activity . Typical marker phenotypes of lymphoid cells observed in the main B- and T-cell LPDs are described; a possibility is mentioned that there can be errors in interpreting the phenotyping results while diagnosing an LPD.

Exp Dermatol, 2003 Oct, 12(5), 700 - 11
CD4 is expressed by epidermal Langerhans' cells predominantly as covalent dimers; Lynch GW et al.; Langerhans' cells (LC) of skin are CD4 expressing, dendritic, antigen-presenting cells, that are essential for activation of primary immune responses and are productively infected by HIV . We have shown previously that lymphocytes and monocytes express CD4 both as monomers and covalently linked homodimers . In those cells the 55-kDa monomer structure predominates . LC in un-fractionated human epidermal cell (EC) suspension also expresses both forms of CD4, but in EC the dimer form is predominant . Because isolation of LC into single cell suspension by trypsin, as is routinely used for LC isolation, degrades CD4, a systematic study for an alternate procedure for LC isolation was performed . Thus it was found that collagenase blend F treatment can efficiently release LC into suspension, under conditions of only minimal degradation of control soluble recombinant CD4 or CEM-T4 or THP-1 cell CD4, or importantly of LC surface CD4 . SDS-PAGE immunoblotting of purified LC extracted from EC by collagenase confirmed CD4 structure as predominantly 110-kDa dimers, with only minimal 55-kDa monomers . The suitability of LC prepared thus for functional studies was demonstrated with binding of functional ligand HIV gp120 . It remains to be determined, however, why tissue embedded LC express mainly CD4 dimers, but single-celled blood lymphocytes and monocytes mainly monomers.

Free Radic Res, 2003 Nov, 37(11), 1163 - 8
The ozone tolerance: I) Enhancement of antioxidant enzymes is ozone dose-dependent in Jurkat cells; Larini A et al.; We have begun to examine the biological and toxic effects of ozone on Jurkat T cells incubated thereafter for 24, 48 and 72 h . Tissue culture medium was strengthened by adding 20% fetal calf serum with an albumin content of about 6 mg/ml . Ozonization was performed by exposing for 10 min a volume of cell suspension (4 x 10(5)/ml) to an equal volume of a gas mixture composed of oxygen-ozone with precise ozone concentrations ranging from 1.5 up to 72 microg/ml (31.5-1512 microM) . The proliferation index declined progressively and was ozone dose-dependent . The response of enzymatic activities varied depending upon the enzyme and ozone concentrations: glucose-6-phosphate dehydrogenase begins to increase at an ozone dose of 6 microg/ml (126 microM), reached a peak at 12 microg/ml (252 microM) and rapidly declined thereafter . On the other hand activities of superoxide dismutase, glutathione peroxidase and glutathione reductase increased progressively from the ozone concentration of 12 microg/ml . Thus, as we have observed in blood, the biological response is linked to the ozone dose that must reach a threshold to be effective.

Zhonghua Yi Xue Za Zhi, 2003 Nov 25, 83(22), 1984 - 8
{Induction of apoptosis in prostate cancer cell line PC-3 by BBSKE, a novel organoselenium compound, and its effect in vivo}; Shi CJ et al.; OBJECTIVE: To investigate the effects of BBSKE (1,2-{bis (1,2-benzisoselenazolone-3 (2H)-ketone)}ethane), a novel organoselenium compound, on the proliferation and apoptosis of the prostate cancer cell line PC-3, and to study its effect on the growth of prostate cancer in vivo . METHODS: Prostate cancer cells of the cell line PC-3 was cultivated in media with different concentrations of BBSKE and cisplatin . The inhibition of proliferation was measured by colorimetric MTT assay . The morphologic changes were observed by fluorescence microscopy, DNA fragmentation was visualized by agarose gel electrophoresis, and the DNA degradation was determined by flow cytometry . Western blot analysis was used to identify the expression of bcl-2 and bax . The activity of caspase-3 was determined by a micro-ELISA reader . Mouse prostate cancer cells of the TRAMP-C2 line were cultured and then injected subcutaneously into 2 male C57BL/6 mice to establish the animal model . Then the 2 mice were killed to collect the cancer cells . Twenty-four mice were injected intraperitoneally with single cell suspension of TRAMP-C2 cell and then divided into 3 groups of 8 mice undergoing intraperitoneal injection for 7 days: BBSKE group (BBSKE was administered at the dosage of 25mg/kg/day), cisplatin group (cisplatin 2mg/kg/d was injected), and control group (pure solvent was injected) . Three weeks after the mice were killed and the tumors were taken out to calculate the inhibition rate . RESULTS: BBSKE inhibited the growth of the PC-3 cells dosage-dependently with a value of IC(50) of 17.90 micro mol/L after a 48 h exposure, higher than that in the case of cisplatin (15.00 micro mol/L) . After exposure of PC-3 cells to BBSKE at the dosage of 20 micro mol/L for 48 hours the apoptosis rate was 26.32%, significantly higher than that of the control group (1.75%, P < 0.01) . The expression of bcl-2 was decreased and the expression of bax remained almost unchanged along with the increase of BBSKE concentration . The activity of caspase 3 in the subgroup of BBSKE of the concentration of 5 micro mol/L remained almost unchanged, and was increased to 3.65 +/- 0.57 and 4.39 +/- 1.01 respectively in the BBSKE 10 micro mol/L and 20 micro mol/L subgroups, both significantly higher than that of the control group (both P < 0.05) . In the in vivo experiment, the growth of tumor was significantly inhibited by BBSKE with an inhibition rate of 40% and the inhibition rate of the cisplatin group was 48% . CONCLUSION: The novel organoselenium BBSKE inhibits the proliferation of PC-3 cell and promote its apoptosis, probably through downregulating the expression of bcl-2 and the activity of caspase-3 . BBSKE also inhibits the growth of prostate cancer in vivo.

Zhonghua Yi Xue Za Zhi, 2003 Dec 10, 83(23), 2067 - 72
{Peroxisome proliferation-activated receptor-gamma ligands ameliorate autoimmune myocarditis associated with inhibition of T cell immunity}; Yuan ZY et al.; OBJECTIVE: To investigate the role of peroxisome proliferator-activated receptor-gamma (PPAR gamma) on autoimmune myocarditis, and to test the hypothesis that PPAR-gamma ligands reduce experimental autoimmune myocarditis (EAM) associated with inhibition of the expansion and activation of self-sensitive T cells . METHODS: EAM was induced in Lewis rats by immunization with porcine cardiac myosin . Then the rats were divided into 3 groups of 9 rats: PPAR-gamma ligand 15-deoxy-(12,14)-PGJ(2) (15d-PGJ(2)) group (15d-PGJ(2) was injected intraperitoneally at the dosage of 200 microg.kg(-1).d(-1)), pioglitazone (PIO) group (PIO was mixed with the food and than fed at the dosage of 10 mg.kg(-1).d(-1)), and positive control group (phosphate-buffered saline was injected intraperitoneally) . Nine normal rats were used as normal controls . Three weeks later, the rats underwent thoracotomy to undergo pathologic examination . The numbers of CD4(+) cells, CD8(+) cells, and macrophages were calculated by microscopy . Immunohistochemistry was used to examine the location and expression of PPAR gamma . Western blotting was used to examine the relative amount of PPAR gamma protein . The proliferative response and the cytotoxicity of T cell-enriched splenocytes and lymph node cells were determined . Another rats were killed 12 days after immunization . Their spleens and lymph nodes were taken out . T-cell rich splenocytes and cells from the lymph nodes were cultured . Cardiac myosin and 15d-PGJ(2) were added . {(3)H} thymine was added 72 hours after . ELISA was used to examine the interferon-gamma (IFN-gamma) in the supernatant . 15d-PGJ(2), PIO, or PBS were given to immunize the rats . The rats were killed 12 days after . The lymph nodes were taken out to make single cell suspension . (51)Cr was used to label the cells so as to calculate the %cytotoxicity . RESULTS: All immunized rats showed myocarditis . The numbers of CD4(+) cells, CD8(+) T cells, and macrophages, were 18 +/- 5, 7 +/- 2, and 45 +/- 8/six 0.25 mm x 0.25 mm squares . PPAR gamma was mainly located in the nuclear and perinuclear regions of infiltrating inflammatory cells, such as mononuclear cells and macrophage-like cells . The expression of PPAR gamma in the myocardium of EAM rats was 3.7 times higher than of the normal rats . The heart weight/body weight ratio, pericardial effusion scores, macroscopic scores and microscopic scores of the 15d-PGJ group were significantly lower than those of the positive control group . The numbers of CD4(+) cells of the 15d-PGJ and PIO groups were 8 +/- 2 and 10 +/- 3, both significantly lower than that of the positive control group (both P < 0.01), the numbers of CD8(+) cells of the 15d-PGJ and PIO groups were 3 +/- 1 and 4 +/- 2 respectively, both significantly lower than that of the positive control group (P < 0.01 and P < 0.05), and the numbers of macrophages of the 15d-PGJ and PIO groups were 22 +/- 4 and 26 +/- 6 respectively, both significantly lower than that of the positive control group (both P < 0.01) . The myocardiogenicity and the severity of myocarditis of the 15d-PGJ(2)- and PIO-groups were at lower degrees compared with those of the positive control group . The % cytotoxic activity was 10.2% +/- 2.6% in the 15d-PGJ(2) group and was 11.6% +/- 3.7% in the PIO group, both significantly lower than that of the positive control group (37.7% +/- 8.4%, both P < 0.01) Stimulated by cardiac myosin, the T-cell rich splenocytes and cells from lymph nodes showed obvious proliferation and production of IFN-gamma . The cardiac myosin-stimulated cell proliferation and production of IFN-gamma in the 15d-PGJ(2) and PIO groups were significantly reduced in comparison with those in the positive control group . CONCLUSION: PPAR-gamma ligands ameliorate EAM associated with inhibition of expansion and activation of the self-sensitive T cells.

Bull Tokyo Dent Coll, 2003 Aug, 44(3), 141 - 7
Effects of a mixed infection with Porphyromonas gingivalis and Treponema denticola on abscess formation and immune responses in mice; Washizu M et al.; Porphyromonas gingivalis and Treponema denticola have been found together in lesions of human periodontitis . We examined the ability of a mixed infection by both bacteria to synergistically form abscesses and disturb immune responses in mice . Absorbance of an invasive P . gingivalis 16-1 strain grown in tryptic soy broth and T . denticola ATCC 33520 strain grown in TYGVS medium were adjusted . BALB/c mice were injected with 200 microliters of the cell suspension at a site on the lateral dorsal area . The sizes of the subsequent subcutaneous abscesses were measured with a caliper gauge, and the area was expressed in square mm . Mixed infections by P . gingivalis and T.denticola produced larger abscesses than those formed after mono-infections by either P . gingivalis or T.denticola . The abscesses caused by mixed infection reached their maxima on the 6th day and maintained that size for the subsequent 5 days . The delayed type hypersensitivities against extracted antigens of P.gingivalis in mixed infection mice were significantly lower than those in the mono-infected mice . However, the IgG response to sonicated antigen of P.gingivalis did not differ between the two groups . The sizes of the abscesses caused by mixed infections in mice immunized with whole cells of P.gingivalis 16-1 were compared to those caused in sham-immunized mice . The average size of the abscess caused by mixed infection in immunized mice did not differ from that in sham-immunized mice, but many of the abscesses in immunized mice ruptured on the 4th or 5th day, followed by recovery in two weeks . These results suggest that mixed infection with P.gingivalis and T.denticola attenuates protective immune responses.

Invest Ophthalmol Vis Sci, 2004 Jan, 45(1), 267 - 74
Transplantation of Schwann cell line clones secreting GDNF or BDNF into the retinas of dystrophic Royal College of Surgeons rats; Lawrence JM et al.; PURPOSE: To assess the capacity of a retrovirus-engineered Schwann cell line (SCTM41), transfected with either a glial cell line-derived neurotrophic factor (GDNF) construct or a brain-derived neurotrophic factor (BDNF) construct, to sustain visual function in the dystrophic Royal College of Surgeons (RCS) rat . METHODS: Cell suspensions were injected into the subretinal space of the right eye of 3-week-old dystrophic RCS rats through a transscleral approach . The left eye remained as an unoperated control . Sham-surgery animals received injections of carrier medium plus DNase to the right eye . All animals were placed on oral cyclosporine . At 8, 12, 16, and 20 weeks of age, animals were placed in a head-tracking apparatus and screened for their ability to track square-wave gratings at various spatial frequencies (0.125, 0.25, and 0.5 cyc/deg) . At the end of the experiment, the animals were perfused and processed for histologic assessment of photoreceptor survival . RESULTS: Animals with SCTM41-GDNF-secreting cells, on average, head tracked longer than animals with SCTM41-BDNF-secreting cells, and both performed better than those injected with the parent SCTM41 line . All tracked longer than sham-surgery or nonsurgical dystrophic eyes . Each cell type demonstrated preservation of photoreceptors up to at least 4 months of age, over and above the sham-surgery control . CONCLUSIONS: Engineered Schwann cells sustain retinal structure and function in the dystrophic RCS rat . Cells overexpressing GDNF or BDNF had a greater effect on photoreceptor survival than the parent line or sham surgery . This study demonstrates that ex vivo gene therapy and subsequent cell transplantation can be effective in preserving photoreceptors from the cell death that normally accompanies retinal degeneration.

J Immunol, 2004 Jan 1, 172(1), 349 - 55
A functional C5a anaphylatoxin receptor in a teleost species; Holland MC et al.; The anaphylatoxins are potent, complement-derived low m.w . proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions . C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes . Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate . To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes . Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression . The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR . Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active . Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a . Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.

J Biochem (Tokyo), 2003 Nov, 134(5), 765 - 72
Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation; Mayer MJ et al.; It was reported recently that overexpression of human ornithine decarboxylase (ODC) cDNA in transgenic rice plants resulted in increased steady-state concentration of polyamines, i.e., enough biosynthetic control is invested at this step to enable adjustment of polyamine levels . To investigate critically whether constitutive overexpression of ODC is sufficient to control steady-state polyamine levels, we expressed an ODC cDNA from Datura stramonium in transgenic tobacco plants . Transgenic progeny of self-fertilised primary transformants exhibited increases in ODC activity of 25-fold in leaves and 5-fold in flower buds . However, the increase in putrescine levels was only 1.5- to 2.1-fold in leaves and 1.1- to 1.3-fold in flower buds . Emphatically, no changes to spermidine or spermine steady-state levels or to soluble or insoluble hydroxycinnamic acid-conjugated polyamines were observed . Ornithine feeding to cell suspension cultures derived from the transgenic plants indicated that putrescine accumulation was limited in part by ornithine availability . These results demonstrate that a large increase in the capacity of the tobacco plants to decarboxylate ornithine does not result in a comparable increase in the level of free or conjugated polyamines . Plant polyamine homeostatic mechanisms efficiently accommodate increased ODC activity, suggesting that polyamine biosynthetic control is invested at multiple interdependent steps.

Life Sci, 2004 Jan 16, 74(9), 1119 - 26
Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study; Vigodner M et al.; The number of cells in the S-phase fraction of the cell cycle reflects proliferative activity . Using flow cytometry histograms and the Phoenix M+ cell cycle program, the percent of cells in the S-phase fraction was measured in single cell suspensions prepared from testes of hamsters of different ages . A cyclical pattern with a period of 9 days, superimposed on another rhythm with a 38 day period was observed (p < 0.01) during hamster maturation and it disappeared after the second spermatogenic wave, where the S phase values reached a plateau . It was concluded that maturing animals passed through a stage in which testicular biological rhythm was involved . Therefore it was concluded that it takes approximately two spermatogenic waves before the proliferation rate in the testis reached a steady state.

Plant Physiol, 2004 Jan, 134(1), 492 - 501 Epub 2003 Dec 18.
Salicylic acid is an uncoupler and inhibitor of mitochondrial electron transport; Norman C et al.; The effect of salicylic acid (SA) on respiration and mitochondrial function was examined in tobacco (Nicotiana tabacum) suspension cell cultures in the range of 0.01 to 5 mm . Cells rapidly accumulated SA up to 10-fold of the externally applied concentrations . At the lower concentrations, SA accumulation was transitory . When applied at 0.1 mm or less, SA stimulated respiration of whole cells and isolated mitochondria in the absence of added ADP, indicating uncoupling of respiration . However, at higher concentrations, respiration was severely inhibited . Measurements of ubiquinone redox poise in isolated mitochondria suggested that SA blocked electron flow from the substrate dehydrogenases to the ubiquinone pool . This inhibition could be at least partially reversed by re-isolating the mitochondria . Two active analogs of SA, benzoic acid and acetyl-SA, had the same effect as SA on isolated tobacco mitochondria, whereas the inactive p-hydroxybenzoic acid was without effect at the same concentration . SA induced an increase in Aox protein levels in cell suspensions, and this was correlated with an increase in Aox1 transcript abundance . However, when applied at 0.1 mM, this induction was transient and disappeared as SA levels in the cells declined . SA at 0.1 mM also increased the expression of other SA-responsive genes, and this induction was dependent on active mitochondria . The results indicate that SA is both an uncoupler and an inhibitor of mitochondrial electron transport and suggest that this underlies the induction of some genes by SA . The possible implications of this for the interpretation of SA action in plants are discussed.

Protein Expr Purif, 2003 Nov, 32(1), 10 - 7
Expression of hepatitis B surface antigen in tobacco cell suspension cultures; Sunil Kumar GB et al.; Hepatitis B virus ' s ' gene coding for surface antigen was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively . Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization . Expression levels as determined by ELISA showed maximum expression levels of 2 microg HBsAg gm(-1) fresh weight and 10 ng mL(-1) of spent medium in pHER100 transformed cells . Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells . HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells . The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g mL(-1) . HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties . This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium.

J Invest Dermatol, 2003 Dec, 121(6), 1425 - 32
Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells; Yamada N et al.; We describe a novel culture system for generating large numbers of murine skin-associated mast cells and distinguish their characteristics from bone marrow-derived cultured mast cells . Culture of day 16 fetal skin single cell suspensions in the presence of interleukin-3 and stem cell factor allowed expansion and maturation of mast cells in the presence of stromal cells . The average yield of mast cells after 2 wk was 7.3 million cells per fetus at a purity of 96% . These fetal skin-derived cultured mast cells increased their histamine content in a time-dependent manner to 3.6 pg per cell after 2 wk and 6.7 pg per cell after 4 wk . Phenotypic analyses revealed much greater expression of CD49b and CD81 and lesser expression of CD77 and CD102 on fetal skin-derived cultured mast cells as compared with bone marrow-derived cultured mast cells . These findings suggest a close similarity between fetal skin-derived cultured mast cells and freshly isolated cutaneous mast cells . Connective tissue mast cell characteristics of fetal skin-derived cultured mast cells were evidenced by: (1) their greater histamine content than bone marrow-derived cultured mast cells; (2) the presence of heparin; and (3) their degranulation in response to compound 48/80 and substance P . Importantly, fetal skin-derived cultured mast cells secreted greater amounts of interleukin-13 but much less MIP-1beta and interleukin-6 than bone marrow-derived cultured mast cells in response to ionomycin . Thus fetal skin-derived cultured mast cells have many characteristics distinct from bone marrow-derived cultured mast cells and can be used as a model of cutaneous mast cells to discern their functions.

Plant Physiol, 2003 Dec, 133(4), 2029 - 39 Epub 2003 Dec 11.
Isolated durum wheat and potato cell mitochondria oxidize externally added NADH mostly via the malate/oxaloacetate shuttle with a rate that depends on the carrier-mediated transport; Pastore D et al.; We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH . We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle . In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane . No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the alpha-glycerophosphate/dihydroxyacetonphosphate shuttles . We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 microm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively) . The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.

Probl Tuberk Bolezn Legk, 2003, (10), 53 - 6
{Activating effect of bestim on the macrophages in experimental tuberculosis of varying severity}; Zabolotnykh NV et al.; The synthetic dipeptide bestim was tested for effects on the functional activity of peritoneal macrophages on 400 non-inbred albino mice while simulating generalized tuberculosis of varying severity (classical, acutely progressive, and slowly progressive) . Bestim was shown to have a stimulating effect on the activity of macrophageal phagocytosis of yeast cell suspension . The agent was also found to exert a restorative effect on the absorptive and digestive functions of macrophages during their inhibition during infection and under the influence of long-term (more than a month) etiotropic therapy . Bestim showed an activating effect on the content of extracellular 5-nucleotidase during the classical and acutely progressive course of infection . It was shown to have a modulating effect on the macrophageal generation of superoxide radicals, by enhancing the inhibited HCT activity in acutely progressive infection and by reducing the elevated level of superoxide production in slowly progressive tuberculosis . In slowly progressive tuberculosis, the drug produced a stimulating effect on the adhesive activity of macrophages.

World J Gastroenterol, 2003 Dec, 9(12), 2859 - 62
Influence of liver nonparenchymal cell infusion combined with cyclosporin A on rejection of rat small bowel transplantation; Yang YL et al.; AIM: To investigate the effect of liver nonparenchymal cell infusion combined with cyclosporin A (CsA) on rejection of heterostrain rat small bowel transplantation . METHODS: The liver nonparenchymal cell suspension was prepared by density gradient centrifugation method with Percoll centrifugal solution . Heterotopic small bowel transplantation was performed . Then the rats were divided into four groups . Group one: homogenic transplantation (F344/N-F344/N), group two: allotransplantation (F344/N-Wistar), group three: allotransplantation (F344/N-Wistar) + CsA, with CsA 10 mg/kg(-1)/d(-1) after transplantation, group four: allotransplantation + CsA (F344/N-Wistar) + liver nonparenchymal cell infusion + CsA (F344/N-Wistar), in which recipient Wistar rats had been injected with 2x10(8) F344/N liver nonparenchymal cells 20 days before transplantation, and treated with CsA after transplantation . Finally, the survival time after small bowel transplantation, gross and histopathological examination, and IL-2 levels in serum were observed . RESULTS: The survival time after small bowel transplantation was 7.14 +/- 0.33 d, 16.32 +/- 0.41 d and 31.41 +/- 0.74 d in group 2, 3, and 4, respectively . The survival time was significant longer (P<0.01) in group 4 . The gross and histopathological examination showed that the rejection degree in group 4 was lower than those in groups 2 and 3 . Serum IL-2 level in group 4 was also lower than those in groups 2 and 3 (P<0.01) . CONCLUSION: Liver nonparenchymal cell infusion combined with CsA can prolong the survival time of rat small bowel transplantation, and the anti-rejection effect is good.

Cell Mol Biol Lett, 2003, 8(4), 979 - 89
Culture treatments for enhancing post-thaw recovery of cryopreserved suspension cells of potato cv . Desiree; Sadia B et al.; An efficient and reproducible protocol has been developed for the cryopreservation of cell suspension cultures of the potato (Solanum tuberosum L.) cv . Desiree . An evaluation was made of the effectiveness of different pre-culture and post-thaw treatments on cell growth, as measured by changes in biomass . Cell suspensions were cultured in UM medium supplemented with 0.25, 0.5, 0.625, 0.75 or 1.0 M sucrose prior to cryopreservation . Sucrose-treated cells were harvested from suspension and 0.75 ml packed cell volumes placed in 2 ml capacity polypropylene vials with 0.5 ml of chilled cryoprotectant (glycerol 46.0 g 1(-1), dimethylsulphoxide 39.0 g 1(-1), sucrose 342.0 g 1(-1) proline 5.0 g 1(-1); pH 5.8) . Cells were frozen at -0.5 degrees C min(-1) from 0 to -35 degrees C, held at -35 degrees C for 35 min and stored, for 10 days, in liquid nitrogen (-196 degrees C) . The most effective pre-treatment, in terms of subsequent post-thaw cell viability as assessed by fluorescein diacetate uptake or triphenyltetrazolium chloride reduction, was culture with 0.75 M sucrose . For this treatment, the mean absorbance (490 nm) following triphenyltetrazolium chloride reduction was 88% greater (p < 0.05) than control and 59% greater (p < 0.05) for thawed cells also cultured on supporting filter paper discs.

Anticancer Res, 2003 Sep-Oct, 23(5A), 3761 - 6
A new orthotopic model of human breast cancer in immunocompetent rats; Akla B et al.; BACKGROUND: Breast cancer is the first cause of mortality by cancer in women in North America and Western Europe . For non-estrogen-dependent tumors as well as for metastatic cancers, current therapies are of limited efficacy . Several animal models have been described but they are imperfect as they are poorly representative of what occurs in real tumors . To overcome some of these limitations, we describe a new orthotopic model of estrogen-independent breast cancer in immunocompetent rats treated with cyclosporin A (CysA) . MATERIALS AND METHODS: Injection of cell suspensions of human breast MDA-MB-231 cancer cells into the mammary fat pad or subcutaneous grafts of solid tumor sections were performed on Sprague Dawley rats receiving intraperitoneally 35 mg/kg of CysA . Tumor growth was measured and monitored by scintigraphy using 18FDG and 99mTc-MIBI . RESULTS: In both models, tumors grew well but growth rates were different . Cell suspension injections gave rise to tumors with a higher growth rate and the tumors obtained infiltrated the mammary and giving results closer to human pathology . CONCLUSION: This animal model with injected cells may provide a new adequate tool for studying breast cancer and developing new therapy strategy.

FASEB J, 2004 Feb, 18(2), 335 - 7 Epub 2003 Dec 04.
Remyelination of the nonhuman primate spinal cord by transplantation of H-transferase transgenic adult pig olfactory ensheathing cells; Radtke C et al.; Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies . However, whether OECs display similar properties in the primate model has not been tested so far . In the present study, we thus transplanted highly-purified OECs isolated from transgenic pigs expressing the alpha1,2 fucosyltransferase gene (H-transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord . Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls . This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs . Additional in vitro assays demonstrated 1) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control . This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination.

Oncol Rep, 2004 Jan, 11(1), 73 - 80
Gemcitabine suppresses malignant ascites of human pancreatic cancer: correlation with VEGF expression in ascites; Kuwahara K et al.; It has been reported that vascular endothelial growth factor (VEGF) is a potent angiogenic factor that also has the ability to increase vascular permeability . VEGF plays an important role in the development of malignant ascites in various cancers . Gemcitabine has been prescribed for patients with inoperable human pancreatic ductal carcinoma as a first-line chemotherapy . However, the response rates of patients with malignant ascites who were undergoing systemic chemotherapy were extremely limited . In the present study, we investigated the role of VEGF and the effects of gemcitabine on malignant ascites of human pancreatic ductal carcinoma . As an in vitro assay, the human pancreatic cancer cell line (SUIT-2) was incubated in DMEM supplemented with serially diluted concentrations of gemcitabine for 24 h . The expression levels of VEGF in culture media were assayed using an enzyme-linked immunosorbent assay (ELISA) . As an in vivo assay, a cell suspension (1 x 10(7) cells in 100 microliters PBS) was injected into the intraperitoneal region . The mice were randomly divided into two groups (control and treated with gemcitabine) . The mice were sacrificed four weeks after inoculation, the ascites volume was measured, and the extent of peritoneal dissemination was examined . The expression levels of VEGF and CD31 in peritoneal nodules were examined by immunohistochemistry . In addition, secreted VEGF protein levels were quantified using ELISA . The results show that VEGF levels in the culture medium decreased in response to gemcitabine in a dose-dependent manner . The ascites formation and peritoneal dissemination within mice were suppressed by the treatment with gemcitabine . Immunohistochemical analysis suggested that expression of VEGF and CD31 in peritoneal nodules was suppressed by gemcitabine treatment, and the VEGF protein level in ascites was significantly decreased by gemcitabine (p<0.05) . These results suggest that gemcitabine controls malignant ascites and peritoneal dissemination, either directly or indirectly, via VEGF . Moreover, intraperitoneal administration of gemcitabine may be a useful therapeutic approach for patients with malignant ascites in pancreatic carcinoma.

Tree Physiol, 2004 Jan, 24(1), 55 - 64
Effects of aluminum on organic acid metabolism and secretion by red spruce cell suspension cultures and the reversal of Al effects on growth and polyamine metabolism by exogenous organic acids; Minocha R et al.; In the absence of added Al, the concentration of succinate in cultured red spruce (Picea rubens Sarg.) cells was 15-20 times higher (> 600 nmol g(FW)(-1)) than that of citrate or oxalate and 4-6 times higher than that of malate . Addition of AlCl(3) (effective monomeric Al concentrations of 0.23 and 0.48 mM) to 3-day-old suspension cultures significantly increased cellular succinate concentrations with a concomitant decrease in cellular oxalate concentrations . However, in the medium of Al-treated cell cultures, both succinate and oxalate concentrations were significantly higher than in the medium of cell cultures without added Al, and oxalate concentrations were several times higher than succinate concentrations . Aluminum did not significantly affect the cellular concentrations of malate, ascorbate and citrate, and none of these organic acids was present in detectable quantities in the medium . Exogenous succinate alone or with Al had no effect on cellular free polyamine concentrations or cell mass . Aluminum caused a significant increase in cellular putrescine concentrations . Addition of malate had a positive effect on growth and completely reversed the effects of Al on cell physiology . In contrast, the addition of oxalate and citrate only partly reversed the effects of Al.

Plant Cell Rep, 2003 Dec, 22(5), 312 - 9 Epub 2003 Aug 29.
Changes in the organization of the tubulin cytoskeleton during the early stages of Solanum lycopersicoides Dun . protoplast culture; Tylicki A et al.; Changes in the tubulin cytoskeleton during protoplast culture and plant regeneration of Solanum lycopersicoides Dun . were analyzed using an immunodetection method . Directly after isolation, four groups of protoplasts were distinguished: (1) mononuclear, (2) polynuclear, (3) homogeneous, (4) anuclear . The tubulin cytoskeleton of the protoplasts underwent rearrangements, correlating to the number and structure of cell nuclei in the protoplast . All protoplast groups with the exception of mononuclear were characterized by perturbations in the organization of the tubulin cytoskeleton . Anuclear and homogeneous protoplasts did not have a tubulin cytoskeleton . Polynuclear protoplasts had cortical microtubules, but were not capable of re-forming their original arrangement and did not possess a radial or perinuclear cytoskeleton . Irregularities in microtubule arrangement of these three groups of protoplasts caused their inability to regenerate a cell wall and to divide . Anuclear, polynuclear and homogeneous protoplasts were eliminated from the culture . Mononuclear protoplasts rearranged their cortical microtubules and reestablished the radial and perinuclear tubulin cytoskeleton . Re-formation of the cell suspension and subsequent regeneration of plants occurred exclusively from mononuclear protoplasts, which were able to regenerate cell walls and to divide.

Fetal Diagn Ther, 2004 Jan-Feb, 19(1), 13 - 22
The intracoelomic route: a new approach for in utero human cord blood stem cell transplantation; Noia G et al.; The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined . Twelve ovine fetuses (gestational ages: 40-45 days) received intracoelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells . Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human beta(2)-microglobulin gene . The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry . Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment . Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells . The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry) . Three fetuses obtained after cesarean section at 102 (No . 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively . A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected . In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta(2)-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle . On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation . This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment .

FEBS Lett, 2003 Dec 4, 555(2), 311 - 6
Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus; Kaminaga Y et al.; Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far . The efficiency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration . Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin . The glucoside yield was 2.5 micromol/g fresh weight of the cells at an optimal culture condition . The water solubility of curcumin-4',4"-O-beta-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.

Neurosci Lett, 2003 Dec 15, 353(1), 5 - 8
Role of the nitric oxide/cyclic GMP pathway and ascorbic acid in 3-morpholinosydnonimine (SIN-1)-induced increases in dopamine secretion from PC12 cells . A microdialysis in vitro study; Serra PA et al.; We showed previously, using in vitro microdialysis, that activation of the nitric oxide (NO)/cyclic GMP pathway was the underlying mechanism of exogenous NO-induced dopamine (DA) secretion from PC12 cells . In this study, infusion of the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mM for 60 min) induced a long-lasting decrease in dialysate DA+3-methoxytyramine (3-MT) in dialysates from PC12 cell suspensions . Ascorbic acid (0.2 mM) co-infusion allowed SIN-1 to increase dialysate DA+3-MT . SIN-1+ascorbic acid effects were abolished by Ca(2+) omission . Infusion of high K(+) (75 mM) induced a 2.5-fold increase in dialysate DA+3-MT . The increase was inhibited by SIN-1 co-infusion . Conversely, co-infusion of ascorbic acid (0.2 mM) with SIN-1+high K(+) resulted in a 3.5 fold increase in dialysate DA+3-MT . The L-type Ca(2+) channel inhibitor nifedipine selectively inhibited the DA+3-MT increase pertaining to high K(+), while the soluble guanylate cyclase (sGC) inhibitor 1H-{1,2,4}-oxadiazolo{4,3}quinoxalin-1-one selectively inhibited the increase pertaining to SIN-1 effects . These results suggest that activation of the NO/sGC/cyclic GMP pathway is the underlying mechanism of extracellular Ca(2+)-dependent effects of SIN-1 on DA secretion from PC12 cells . Extracellular Ca(2+) entry occurs through nifedipine-insensitive channels . Ascorbic acid is a key determinant in modulating the distinct profiles of SIN-1 effects.

J Endourol, 2003 Nov, 17(9), 709 - 17
Conversion of an HM3 lithotripter into a research device; Loske AM et al.; PURPOSE: To describe the conversion of a Dornier HM3 lithotripter into a research device and evaluate its performance . MATERIALS AND METHODS: A used HM3 lithotripter was donated to our university by the St . Thomas' Hospital in London . It was disassembled, shipped to our laboratory, partially assembled, and modified as a research lithotripter . Pressure measurements at several positions and kidney stone model fragmentation tests were performed to evaluate the modified system . Results were compared with information published by other authors and data obtained in our laboratory using another electrohydraulic research lithotripter . RESULTS: Pressure records showed typical lithotripter waveforms with a rapid rise to about 50 MPa, followed by decay to a negative peak of approximately 9 MPa . Maximum compressional peaks were obtained at F2 and 25 mm below F2 . Kidney stone model fragmentation was typical for electrohydraulic shockwave lithotripters . CONCLUSIONS: Comparison of pressure measurements with data obtained by other authors on the same lithotripter several years ago indicate that the pressure waveform has not changed significantly . A much smaller water tank, a small X-Y-Z positioner, and no X-ray imaging system facilitate the use of this shockwave generator for in vitro experiments with small samples such as vials containing cell suspensions, having the advantage of a reliable, well-known, and well-characterized commercial shockwave generator.

Cytometry, 1996 Jan 1, 23(1), 82 - 9
Improved kinetic analysis of cytosolic free calcium in pressure-sensitive neuronal cells by fixed-time flow cytometry; Tarnok A; Two flow cytometric techniques were used to measure rapid transient changes in {Ca2+} in the neuronal cell line NH15-CA2 . Using on-line injection, the cell suspension and stimulating solution are mixed and delivered to the detection point by a rapid increase in sample pressure . In NH15-CA2, injection of medium alone resulted in {Ca2+}i increase . Using the fixed-time method, where cells are maintained at constant pressure, no {Ca2+ }i, increase was observed with medium alone . These results show that a rapid pressure increase alone alters the {Ca2+}i in NH15-CA2 cells . Both methods showed similar kinetics of {Ca2+}, in response to bradykinin but the fixed-time method was found to be better for determination of the percentage of responsive cells.

Z Rheumatol, 2003, 62(Suppl 2), II46 - 9
{Tissue engineering: chances and challenges for application in rheumatic diseases}; Haupl T et al.; Current technologies of tissue engineering offer new strategies for the treatment of cartilage and bone defects . Beyond implantation of cell suspensions, second generation products of biomaterial enforced with in vitro preformed tissues are clinically applied . Ongoing research and development focus on differentiation factors and tissue protection . In search for sources of autologous cells which are easier to collect and which may serve for more complex tissues like osteochondral implants, mesenchymal stem cells are investigated . The design of in vitro experiments, which are required for these investigations, has produced tissue engineering technologies, which may serve for pathophysiology research in inflammatory joint diseases and for exploration of treatment strategies . These together with the advances in biological therapies of rheumatic diseases are the basis of new concepts, which promise application of tissue engineering also in inflammatory joint diseases.

Chemosphere, 2004 Feb, 54(7), 905 - 15
Modelling of the acid-base properties of natural and synthetic adsorbent materials used for heavy metal removal from aqueous solutions; Pagnanelli F et al.; In this paper a comparison about kinetic behaviour, acid-base properties and copper removal capacities was carried out between two different adsorbent materials used for heavy metal removal from aqueous solutions: an aminodiacetic chelating resin as commercial product (Lewatit TP207) and a lyophilised bacterial biomass of Sphaerotilus natans . The acid-base properties of a S . natans cell suspension were well described by simplified mechanistic models without electrostatic corrections considering two kinds of weakly acidic active sites . In particular the introduction of two-peak distribution function for the proton affinity constants allows a better representation of the experimental data reproducing the site heterogeneity . A priori knowledge about resin functional groups (aminodiacetic groups) is the base for preliminary simulations of titration curve assuming a Donnan gel structure for the resin phase considered as a concentrated aqueous solution of aminodiacetic acid (ADA) . Departures from experimental and simulated data can be interpreted by considering the heterogeneity of the functional groups and the effect of ionic concentration in the resin phase . Two-site continuous model describes adequately the experimental data . Moreover the values of apparent protonation constants (as adjustable parameters found by non-linear regression) are very near to the apparent constants evaluated by a Donnan model assuming the intrinsic constants in resin phase equal to the equilibrium constants in aqueous solution of ADA and considering the amphoteric nature of active sites for the evaluation of counter-ion concentration in the resin phase . Copper removal outlined the strong affinity of the active groups of the resin for this ion in solution compared to the S . natans biomass according to the complexation constants between aminodiacetic and mono-carboxylic groups and copper ions.

Cancer Res, 2003 Nov 15, 63(22), 7986 - 94
In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy; Niedre MJ et al.; Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT) . We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo . Here, we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX) . Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10, 25, or 50 mWcm(-2) and, (1)O(2) luminescence measurements were made throughout the treatment . Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay . The PpIX concentration in the cells, the photobleaching, and the pO(2) in the cell suspensions were also monitored . There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation) . However, in all of the cases, cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill . This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT, as well as confirming the likely role of (1)O(2) in porphyrin-based PDT.

Tissue Eng, 2003 Oct, 9(5), 931 - 8
Homogeneous seeding of mesenchymal stem cells into nonwoven fabric for tissue engineering; Takahashi Y et al.; Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rats and seeded into a nonwoven fabric of polyethylene terephtalate (PET) by agitation and static methods . MSC attachment was investigated in terms of the number of cells attached to the fabric, their distribution inside the fabric, and cell damage . The number of MSCs attached was greater for the agitation seeding method than for the static seeding method . The higher the rotating speed in the agitation seeding method, the greater the number of cells attached . When the cell suspension was seeded into the fabric in culture medium volumes of 50 and 200 microL per well of the culture plate or per culture tube, the best cell attachment was observed for the tube culture group at the larger volume . These cells attached more homogeneously throughout the fabric in greater numbers than was the case for the other culture groups . It is possible that agitation of the cell suspension allows cells to infiltrate uniformly inside the fabric, resulting in a homogeneous distribution of the cells in the fabric . A biochemical study revealed that neither the agitation nor static seeding method damaged cells, irrespective of the medium volume and the type of culture vessel . We conclude that the agitation seeding method is a promising method by which to formulate a homogeneous construct of fabric and MSCs.

J Appl Microbiol, 2003, 95(6), 1304 - 14
Development of method to quantify extracellular carbohydrate complexes produced by Escherichia coli O157:H7; Ryu JH et al.; AIMS: The aim of this study was to optimize conditions to separate extracellular carbohydrate complexes (ECC) produced by Escherichia coli O157:H7 and to standardize the amount of ECC produced on a per cell basis . METHODS AND RESULTS: ECC fraction I was removed from E . coli O157:H7 cells produced on tryptic soya agar and lettuce juice agar by centrifugation . To remove ECC fraction II, cells were heated at 100 degrees C for 10 min, then centrifuged . The sum of ECC fractions I and II was considered as the total ECC produced by E . coli O157:H7 . A correlation between cell mass and turbidity (O.D . 750 nm) of cell suspensions was determined . Cell mass has a linear relationship (R2 = 0.93) with turbidity of cell suspensions from which ECC is removed . The amount of ECC produced on a per cell basis was calculated by dividing total amount of ECC (microgram ml-1) produced by the turbidity (O.D . 750 nm) of heated cell suspension after removal ECC fractions I and II . CONCLUSIONS: A method for separating ECC from cells of E . coli O157:H7 has been developed and conditions have been optimized . A standard method to estimate the amount of ECC produced on a per cell basis was also developed . SIGNIFICANCE AND IMPACT OF THE STUDY: Using these procedures to prepare extract of ECC from E . coli O157:H7 and to standardize values, production of ECC on a per cell basis can be estimated and a comparison of the amount of ECC produced by the pathogen grown under different environmental conditions can be accurately measured.

Pigment Cell Res, 2003 Dec, 16(6), 644 - 55
Steel factor controls the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture; Hirobe T et al.; Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF) . Pure cultured primary melanoblasts and melanocytes were then further cultured with MDMD/MDMDF supplemented with steel factor (SLF) (keratinocyte depletion) . SLF increased the number of melanoblasts and melanocytes as well as the proportion of differentiated melanocytes in the absence of keratinocytes . Flow cytometric analysis showed that melanoblasts and melanocytes in the S and G2/M phases of the cell cycle were increased by treatment with SLF . Moreover, an anti-SLF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes . These results suggest that SLF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.

Zentralbl Gynakol, 2003 Oct, 125(10), 409 - 14
{Investigations on regulation of CRH, ACTH and cortisol in trophoblast cells in vitro}; Hocker I et al.; OBJECTIVE: Trophoblast cells synthesise CRH and ACTH, which are peptide hormones . On the strength of special enzymes they are capable of catalyzing the reaction cortisol <--> cortisone . In vitro experiments should give a proof of influence to ACTH- and cortisol secretion by CRH, ACTH and prednisolon . The basal rate of cortisol secretion was examined in a long term experiment . MATERIAL AND METHODS: Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step . After adjusting the cell suspension to a defined cell concentration of 1 x 10(6) cells/ml the cells were cultivated . The addition of CRH, ACTH or prednisolon followed every eight hours . The samples collected 20 or 30 minutes later, also from unstimulated cultures, were assayed for ACTH and cortisol by enzyme-immunometric methods . RESULTS: The concentration of cortisol shows a rhythmical course in long term cell cultures . The addition of CRH (500 ng/ml, 1 microg/ml) stimulates the concentration of ACTH- and cortisol in a time-depending manner . The addition of ACTH (500 ng/ml-2 microg/ml) stimulates the concentration of cortisol in a time-depending manner . The addition of prednisolon stimulates the concentration of ACTH . CONCLUSIONS: The trophoblast cell shows a rhythmical course in the concentration of cortisol . For the first time a CRH-ACTH-cortisol feedback loop could be demonstrated in cultured trophoblast cells.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Oct, 34(4), 736 - 7, 741
{The modified isolation and culture method of rat Sertoli cells}; Wang W et al.; OBJECTIVE: To simplify the procedures of isolation of rat Sertoli cells and increase the yield of Sertoli cells cultured . METHODS: Decapsulated rat testis from 16-22-day-old SD rats were digested by sequential two-step digestion using 0.25% pancreatin, 0.05% collagenase in order, and then the cell suspension was cultured in incubator under the condition of 37 degrees C and 5% CO2 for 48 h . The yields were observed and Fas Ligand (Fas-L) was tested by immunohistochemical method . RESULTS: By the use of this modified method, the yielded sertoli cells accounted for 90% of all the cells harvested . High level expression of Fas-L was detected . CONCLUSION: This modified method increases the yield of Sertoli cells and the procedures of isolation and culture are greatly simplified.

ANZ J Surg, 2003 Nov, 73(11), 894 - 6
Decreased phagocytic capacity of autotransplanted splenic tissue; Smith E et al.; BACKGROUND: Asplenic patients have an increased risk of infections . Operations such as autotransplantation have been proposed to restore functional splenic tissue after splenectomy, but the protective value of this tissue is unclear . Immune responses such as production of antibody remain impaired in humans and animals even when such tissue is present, and clearance of particles from the blood is reported to be less efficient than by normal spleen tissue . The present study investigated the phagocytic capacity of cells in the regenerated tissue in vitro, free of the confounding effects of hepatic clearance . METHODS: Single cell suspensions were prepared from splenic tissue from rats 6 months after splenic autotransplantation or sham operation . Phagocytosis of killed, fluorescein-labelled bacteria was measured by flow cytometry . RESULTS: Autotransplanted tissue contained fewer phagocytic cells than normal tissue, and these cells phagocytosed less per cell . Phagocytosis by spleen cells was dependent on heat-labile opsonic factors . CONCLUSIONS: Autotransplanted splenic tissue does not restore the phagocytic capacity lost following splenectomy.

J Vet Sci, 2001 Apr, 2(1), 43 - 6
Flow cytometric evaluation on the age-dependent changes of testicular DNA contents in rats; Yoon CY et al.; An age-dependent cellular change of DNA contents in the testis of Sprague-Dawley rats was investigated by flow-cytometric method . Testicular cell suspensions at the age of 4, 5, 6, 7, 8, 10, 12, 16 and 26 weeks were prepared and stained with propidium iodide . The relative proportions in the number of mature and immature haploid (1n), diploid (2n), S-phase and tetraploid (4n) cells were calculated . The proportion in the number of mature haploid cells was sharply increased to the age of 10 weeks (about 38%), thereafter increased slightly to the level of 42% at the age of 26 weeks . The proportion of immature haploid cells was dramatically increased to the age of 6 weeks, then maintained at the level of 20 to 30% thereafter . The proportion of diploid cells was 64% at the age of 4 weeks, then decreased gradually through the age of 26 weeks . The proportion of S-phase cells was increased to the age of 4 weeks, then maintained at a plateau level to the age of 26 weeks . The proportion of tetraploid cells were about 26% at the age of 4 weeks, then decreased gradually to the age of 26 weeks . These results suggest that the proportions of testicular cells may depend on the age of the rat and that the flow cytometric method may be useful in the evaluation of the spermatogenic status with regard to accuracy and sensitivity.

Brain Res Brain Res Protoc, 2003 Oct, 12(2), 67 - 76
Purification of astrocytes from adult human optic nerve heads by immunopanning; Yang P et al.; Most in vitro studies in the CNS require pure cultures of astrocytes . Astrocytes from the human optic nerve head (ONH, type 1B) represent a specialized population of astrocytes . Primary cells grown from human optic nerve head explants were cultured for 3-4 weeks . To select astrocytes by immunopanning, cell suspensions were placed on a P100 panning dish coated with C5 anti-neuroepithelial antibody and allowed to attach for 30 min . Nonadherent cells were plated on a second dish coated with anti-Thy1.1 antibody to deplete microglia and meningeal cells . Finally, remnant nonadherent cells were plated on a noncoated dish . Purified cells were immunostained with astrocyte markers: GFAP, vimentin, Pax2, A2B5, nestin and NCAM . Other cell types were characterized by HLA-DR for microglia and smooth muscle actin for vascular smooth muscle . The proportion of GFAP+ astrocytes in the cultures was determined by flow cytometry . About 95% of the cells that adhered to the C5 dish were GFAP+ astrocytes . GFAP+ astrocytes expressed vimentin, Pax2, nestin and NCAM, but not A2B5 . From the Thy1.1 dish, 60-75% cells were GFAP+ astrocytes and the remainder cells were GFAP- cells . Using cloning rings, we eliminated fibroblast-like cells, smooth muscle and meningeal cells from astrocyte cultures . Smooth muscle cells and fibroblasts grew on the noncoated dish . In conclusion, immunopanning is an efficient method to get high yields of viable type 1B astrocytes from adult human ONH . The current described culture system may provide a valuable tool in studying human optic nerve head biology and disease.

J Biomed Mater Res A, 2003 Dec 1, 67(3), 944 - 51
Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model; Sikavitsas VI et al.; The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site . Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups . A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats . Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response . Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups . Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups) . Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%) . They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation . There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group . However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site . They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential .

Immunol Lett, 2003 Nov 15, 90(1), 13 - 8
Comparison of CTL reactivity in the spleen and draining lymph nodes after immunization with peptides pulsed on dendritic cells or mixed with Freund's incomplete adjuvant; Wang MJ et al.; OBJECTIVE: To compare CTL reactivity in the spleen and the draining lymph nodes (LN) from C57BL/6 mice after immunization with self and non-self peptides pulsed on autologous dendritic cells (DC) or mixed with Freund's incomplete adjuvant (FIA) . METHODS: Peptides showing high to low binding affinities for H-2 Kb/Db were emulsified in FIA or pulsed on bone marrow (BM)-derived DC and injected subcutaneously into C57BL/6 mice . Eight days later, the mice were sacrificed and cell suspensions were prepared from the spleen and draining LN . Splenocytes or LN cells were cultured for 5 days with irradiated syngeneic spleen cells (as APCs) pulsed with the appropriate peptide in vitro . 51Cr-release assay using peptide pulsed target cells was used to detect CTL reactivity . RESULTS: Both self and non-self peptides can induce specific CTL responses with the adjuvant FIA and DC . Peptide pulsed DC were found to be more effective than peptides mixed with FIA to induce specific CTL responses towards non-self peptides and can induce much stronger responses in the spleen than in the draining LN both for non-self and self peptides . Self peptides emulsified in FIA generated the strongest responses in the draining LN, whereas non-self peptides mixed with FIA generated the strongest response in the spleen . CONCLUSIONS: DC-based immunization with non-self and self peptides is more efficient than immunization based on peptides mixed with FIA . DC-based immunization focuses the CTL response towards the spleen . Immunization based on FIA focuses the response against self peptides towards the draining LN and non-self peptides towards the spleen.

Reproduction, 2003 Nov, 126(5), 599 - 604
Spermatogenesis in testes of Dazl null mice after transplantation of wild-type germ cells; R R et al.; Dazl knockout male mice are infertile because their germ cells are unable to complete the first meiotic prophase in the first wave of spermatogenesis and thereafter decrease in number due to a block at the A-aligned to A1 transition . The ability of the surviving somatic components of the testes to retain their function in the absence of mature germ cells was tested by injecting marked wild-type germ cell suspensions containing spermatogonial stem cells . Comparison of the frequency and extent of colonization of Dazl knockout testes with that of testes chemically depleted of germ cells showed little if any difference . It was concluded that Dazlko testes seem unimpaired in their ability to support spermatogenesis . Therefore, Dazlko testes provide a useful and reliable recipient in which to evaluate spermatogonial stem cells . The results furthermore demonstrate that the somatic compartment of the testis of these animals retains functionality.

Am J Clin Pathol, 2003 Nov, 120(5), 754 - 9
Diagnostic significance of CD20 and FMC7 expression in B-cell disorders; Delgado J et al.; We analyzed by flow cytometry the expression of CD20 and FMC7 in cell suspensions from 932 patients, including 630 cases of chronic lymphocytic leukemia (CLL), 23 cases of other B-cell leukemias, and 279 cases of B-cell non-Hodgkin lymphoma (B-cell NHL) . CD20 was positive in 94.5% of cases; FMC7 was positive in 35.7% . There was a correlation between CD20 and FMC7 expression in patients with B-cell NHL (P < .001) but not CLL (P = .1) . We also tested a scoring system in which FMC7 was replaced by CD20 and compared it with our current scoring system for CLL . With this modification, the accuracy of the scoring system for differentiating CLL from other non-CLL disorders fell from 94.4% to 81.5% . In CD20+ CLL, the intensity of CD20 expression correlated with FMC7 and low scores (P < .001 for both comparisons) . We suggest that the particular conformation of CD20 recognized by FMC7 is manifested only in cells with strong CD20 expression, which is not the case for CLL . FMC7 is of greater diagnostic value than CD20 for distinguishing CLL from other B-cell disorders; we recommend its continued use for this purpose.

J Cancer Res Clin Oncol, 2004 Jan, 130(1), 45 - 51 Epub 2003 Nov 07.
Enhancement of effects of irradiation by gemcitabine in a glioblastoma cell line and cell line spheroids; Genc M et al.; BACKGROUND AND PURPOSE: To determine the cytotoxicity of, and radioenhancement by, gemcitabine on a glioma cell line grown as a monolayer and as spheroid cultures . MATERIAL AND METHODS: We used a human glioma cell line, Gli-6, which originated from a biopsy specimen of a patient with a glioblastoma multiforme . Spheroids of Gli-6 were prepared by seeding a single cell suspension on agarose-coated Petri dishes . Clonogenic and growth delay assays were used to determine radio-chemosensitivity of monolayer cultures . The growth delay assay was used to determine that of Gli-6 spheroid cultures . RESULTS: Spheroid cultures were found to be more resistant to irradiation with/or without gemcitabine than monolayer cultures . Whereas gemcitabine significantly enhances the radiation effect of exponentially growing Gli-6 monolayer cultures at minimal cytotoxic concentrations (10 nM, 24 h), no enhancement was seen in confluent monolayer cultures and in large spheroids at the same concentration . In small spheroids no enhancement was observed at a low-dose gemcitabine (10 nM for 24 h), but an enhancement was observed at higher concentrations (100 nM for 24 h) . CONCLUSION: Gemcitabine can lead to enhancement of the effects of X-irradiation in both monolayer as spheroid glioblastoma cultures . The lack of enhancement in confluent monolayer cultures supports the view that cell cycle distribution of cells is important in radiosensitisation by gemcitabine

Brain Tumor Pathol, 2003, 20(1), 1 - 5
Reduction of nitroxides and radioprotective ability in glioblastoma cells; Moritake T et al.; Electron spin resonance (ESR) analyses were performed to clarify whether glioblastoma cells scavenge hydroxyl radicals (*OH) generated by x-ray irradiation . The rate of bioreduction of nitroxides by three human glioblastoma cells was also evaluated by the same technique and compared with their x-ray sensitivity . Aerated culture media containing 200mM of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with or without U87MG cells were irradiated with x-rays at a dose of 20Gy . ESR was measured immediately after each irradiation . Continuous changes of the ESR spectra of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) were analyzed in cell suspensions of TK1, U87MG, and A172 at a concentration of 1.0 x 10(7) cells/ml containing 5 microM Tempol . As a result, the signal of DMPO-OH in the U87MG cell suspension decayed faster than that in the control culture media without cells, and the rate of bioreduction of Tempol in each glioblastoma cell suspension was correlated with the x-ray sensitivity defined from the colony-forming assay in those cell lines . It was indicated that the resistance of glioblastoma cells to ionizing radiation could be closely related to their ability to scavenge radical species generated by ionizing radiation.

J Cell Physiol, 2004 Feb, 198(2), 209 - 22
Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways; Harnois C et al.; To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model . Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase {MAPK} kinase (MEK)/extracellular regulated kinases (Erk) pathways . Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension . The activation levels of studied kinase pathways were also analyzed . Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells . Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells . However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied . For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed . These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death . In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs .

Ann Acad Med Stetin, 2002, 48, 117 - 33
{Freezing of umbilical blood cells in mechanical freezers (-80 degrees C)}; Paczkowska E; Umbilical cord blood (UCB) contains hematopoietic stem cells capable of reconstituting hematopoiesis in the recipient . This property has prompted search for methods to store such cells in liquid nitrogen (-196 degrees C) and offer them for future use in HLA-matched patients . UCB cells are often collected in hospitals remote from storage facilities . Immediate freezing is important to prevent apoptosis which develops relatively quickly after isolation from cord blood . Investigations into the freezing of bone marrow cells have corroborated the feasibility of uncontrolled rate freezing and storage of cells in a mechanical freezer at -80 degrees C . The aim of the present study was to apply this approach to UCB cells and to determine optimal freezing conditions as measured by progenitor cell survival . The highest survival rate among UCB cell species subjected to mechanical freezing was demonstrated by mononuclear cells enriched for the CD34+ subpopulation . The presence of CD34- mononuclear cells (monocytes, lymphocytes) and erythrocytes in the cell suspension reduced progenitor cell survival and proliferation potential . Moreover, it was found that CD34+ cells from UCB can be frozen in an uncontrolled manner . Optimal results were achieved with 10% DMSO and 70% serum . The results were unaffected by the type of medium used (RPMI, Dulbecco, Iscove) . In conclusion, uncontrolled rate freezing of UCB cells is an acceptable method prior to transport of the cells in dry ice to a central banking station and storage in liquid nitrogen until needed.

Toxicology, 2003 Dec 1, 193(3), 269 - 79
A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies; Marcusson-Stahl M et al.; A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies . The classical 51Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems.Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development.The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes . The results are expressed as percentage of dead targets on a cell-to-cell basis.The final format of the assay includes 0.5ml peripheral blood, 1.25x10(5) effector cells per sample, and collection of 500 target events by flow-cytometry . When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08% . The corresponding cytotoxic activity decreased from 68 to 8% . Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension . Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells.The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies . Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled.

Electrophoresis, 2003 Oct, 24(19-20), 3421 - 32
Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis; Borderies G et al.; The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs) . Most proteomic approaches depend on the quality of sample preparation . Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked by covalent bonds . Specific and sequential extraction procedures thus need to be developed . We report on the sequential extraction of loosely bound CWPs from living A . thaliana cells in culture . Different salts and chelating agents were used for releasing the proteins from the wall . Their effects on the extraction of CWPs and on the integrity of the plasma membrane were evaluated . Bioinformatic software was used to identify proteins and to predict their sub-cellular localization . The obtained data show that the plasma membrane of cells in culture was easily damaged by some steps of the extraction procedure, leading to the release of increasing amounts of intracellular proteins . Nevertheless, we identified fifty CWPs among which thirteen were new proteins for the cell wall . In addition, 76% of these CWPs were basic proteins not resolved in two-dimensional (2-D) gel electrophoresis . The existence of two hypothetical proteins was confirmed . The structure of three proteins could be confirmed using mass spectrometry data.

Stem Cells, 2003, 21(6), 638 - 46
A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity; Noia G et al.; The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined . Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells . Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin . Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment . Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells . The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry) . Three fetuses obtained after cesarean section at 102 (no . 435184) and 105 (no . 915293, no . 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively . In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle . This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13152 - 7 Epub 2003 Oct 31.
Regulation of CD18 expression on neutrophils in response to fluid shear stress; Fukuda S et al.; When leukocytes adhere to endothelial cells and are exposed to fluid shear stresses, they often retract pseudopods and reduce their attachment . Leukocytes use CD18 for membrane adhesion, but the kinetics of such integrin adhesion molecules under fluid shear is unknown . We examine on neutrophils with confocal microscopy of single adherent cells and flow cytometry of cell suspensions the CD18 expression under fluid shear after labeling with fluorescent antibodies . Fluid shear causes reduction of CD18-associated immunofluorescence of extracellular epitopes, especially in areas of the membrane exposed to elevated levels of shear (1.5 dyne/cm2 maximum shear stress; 1 dyne = 10 mN) . CD18 was also translocated over the leukocyte surface from regions of higher shear to lower shear and into the membrane contact areas with the substrate . We obtained no evidence for cytoplasmic internalization of CD18 . Fluid shear (5 dyne/cm2) in a suspension of human leukocytes resulted in cleavage of the extracellular domain but not against a cytoplasmic domain of CD18 . Chelation of extracellular Ca2+ abolished the down-regulation of CD18 . Cysteine protease inhibitors and a selective inhibitor for cathepsin B, but no blockade of other cysteine proteases such as cathepsin L and calpain, aminopeptidases, elastase, or metalloproteinases, suppressed shear-induced CD18 down-regulation . The evidence suggests that physiological levels of fluid shear cause release of cysteine protease(s) including cathepsin B, leading to cleavage of the extracellular domain of CD18 molecules and possible membrane detachment.

J Hematother Stem Cell Res, 2003 Oct, 12(5), 515 - 23
Large-scale immunomagnetic selection of CD14+ monocytes to generate dendritic cells for cancer immunotherapy: a phase I study; Babatz J et al.; Dendritic cells (DC) are professional antigen-presenting cells that are widely used in the experimental immunotherapy of cancer . For clinical use GMP-like protocols for the preparation of functionally active dendritic cells (DC) in large numbers and at high purity are needed . However, the currently available protocols have certain disadvantages . In this study we tested the generation and clinical applicability of DC from monocyte preparations produced by immunomagnetic CD14(+) selection using a semiautomated clinical scale immunomagnetic column . Peripheral blood mononuclear cells (PBMC) of 10 patients with metastatic solid tumors were used . With the immunomagnetic separation, we obtained a cell suspension of high CD14(+) purity (median 97.4%, range 94.9-99.0) with a high monocyte yield (median 82.3%, range 63.9-100.0) . Differentiation of CD14(+) cells into mature monocyte-derived DC was induced by incubation with IL-4, GM-CSF, TNF-alpha, PGE(2), IL-1 beta, and IL-6 . Mature DC showed a high expression of CD83, HLA-DR, and the co-stimulatory molecules CD80 and CD86 . Overall CD83(+) yield was 12.1% (range 4.0-29.4) . Allogeneic T stimulatory capacity could be demonstrated for all DC preparations in proliferation assays . No significant differences in marker expression or T cell stimulation was detected between fresh DC and those derived from cryopreserved immature DC . Clinical administration of autologous DC by three different parenteral routes was tolerated by all 10 patients without systemic signs of toxicity . Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS device is a suitable method for clinical-scale generation of functional DC under GMP-grade conditions . The selection can be performed in a closed system . Therefore, immunomagnetic CD14(+) selection can be seen as an alternative way to generate DC for clinical tumor vaccination protocols.

J Plant Physiol, 2003 Sep, 160(9), 1117 - 24
Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca; Bhuiyan NH et al.; Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors . Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan) . Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively . A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ) . MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3 . However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA . Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L) . The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied . The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan . Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis . This pathway seems to act antagonistically towards beta-glucan-mediated signaling . As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.

J Plant Physiol, 2003 Sep, 160(9), 1025 - 32
Determination of phenolic antioxidant compounds produced by calli and cell suspensions of sage (Salvia officinalis L.); Santos-Gomes PC et al.; Sage (Salvia officinalis L.) calli were established by culturing internodal segments, excised from aseptic seedlings, on MS basal medium gellied with agar and supplemented with 0.05 mg/L dichlorophenoxyacetic acid (2,4-D) in presence of benzyladenine (BA) or zeatin (ZEA) or kinetin (KIN), at 1.5 mg/L . Suspended cells were established by transferring one callus to 50 mL of liquid MS basal medium devoid of agar and containing the same type of hormonal supplementation used in respective calli growth . The highest growth of calli and suspensions occurred with 1.5 mg/L ZEA . However, with this cytokinin supplementation, as well as with 1.5 mg/L KIN, both in presence of 0.05 mg/L 2,4-D, suspensions differentiated small root shaped structures . Well shaped, majority single cell suspensions were formed under the effect of 0.05 mg/L 2,4-D and 0.5 mg/L KIN . Calli grown with 0.05 mg/L 2,4-D and 1.5 mg/L BA and suspended cells grown with 0.05 mg/L 2,4-D and ZEA or KIN at 1.5 mg/L, or KIN at 0.5 mg/L, were searched for phenolics production . Twelve phenolic compounds were identified in calli: gallic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, rosmarinic acid, hesperetin, epirosmanol, hispidulin, genkwanin, carnosol, carnosic acid, and methyl carnosate . With the exception for genkwanin and epirosmanol all of these phenolic compounds were also produced by the sage suspension cultures grown in the presence of 1.5 or 0.5 mg/L KIN . Genkwanin was the only phenolic absent in the suspensions grown with 1.5 ZEA . Suspended cells, grown with 0.5 mg/L KIN, and calli cultures showed the highest specific accumulation of the total phenolics, with rosmarinic acid representing 94-97%.

J Mol Neurosci, 2003, 21(2), 111 - 20
Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling; Junger H et al.; Extracellular hypertonicity can induce the phosphorylation of mitogen-activated protein kinases (MAPKs) . Of these, both extracellular signal-regulated kinases (ERKs) and the stress-activated kinase p38 have been implicated in neuronal cell survival . Resuscitation with hypertonic saline decreases secondary brain injury after trauma, as well as neuronal damage, after ischemia . Since hypertonicity has been shown to support somatic cell survival, we investigated if hypertonicity can also prevent neuronal cell death via MAPK signaling . Death of postnatal rat corticospinal motoneurons (CSMNs) was induced by serum deprivation, and survival in both isotonic and hypertonic media was assessed after 20 h . Addition of NaCl (4-250 mM) to isotonic medium significantly and dose dependently protected CSMN in enriched cultures, increasing cell survival by up to 70% over that in isotonic medium . This response was not restricted to NaCl; addition of KCl, choline chloride, and sucrose had similar effects on cell survival . In addition, hypertonicity supported the survival of pure CSMN populations, albeit with lower potency . In cortical cell suspensions, hypertonic NaCl (20-100 mM) increased basal phosphorylation of p38 and ERK . The activation of both MAPKs, which was induced by 40 mM NaCl, was transient . Cultivation of CSMNs in media containing the specific p38 inhibitor SB203580 abolished the protective effect of hypertonic NaCl, indicating a central role for p38 . We therefore conclude that hypertonicity can prevent neuronal cell death via MAPK signaling.

J Bone Miner Res, 2003 Oct, 18(10), 1882 - 8
Characterization of C4-2 prostate cancer bone metastases and their response to castration; Pfitzenmaier J et al.; New well-characterized preclinical models of prostate cancer (CaP) bone metastases are needed to improve our understanding of the development of CaP-related bone disease in patients . Here we describe characterization of a model consisting of direct injection of C4-2 cells into tibias . INTRODUCTION: Prostate cancer (CaP) has a high proclivity to metastasize to bone . Development and characterization of preclinical models of CaP bone metastases are of high interest . The objective of this study was to characterize C4-2 bone metastases and their response to castration . MATERIALS AND METHODS: Cell suspensions of C4-2, a subline of LNCaP, were injected directly into the tibias of intact male mice . In groups A (n = 7) and B (n = 5), animals were killed 3 and 8 weeks after injection of C4-2 cells, respectively . In group C (n = 7), animals were castrated 3 weeks after injection and killed 5 weeks after castration . Serum prostate-specific antigen (PSA) levels and bone mineral density (BMD) were measured, and bone histomorphometric analysis was performed . RESULTS: C4-2 cells decreased BMD of the injected tibias by 36.1% and bone volume by 74.1% versus normal tibias . Castration caused a 32.3% drop in serum PSA (p = 0.0438), with a nadir at day 14, after which it began to rise again . Bone destruction in the tumorous tibias of castrated animals was decreased by 15.9% versus tumorous tibias of intact animals (p = 0.0392) . However, BMD in the tumorous tibias of castrated mice was still lower than in normal tibias of intact animals . Castration also decreased BMD and bone volume in nontumorous tibias (p = 0.0406 and 0.0232, respectively) . CONCLUSIONS: The C4-2 model of bone metastasis recapitulates the response to androgen deprivation observed in CaP patients with bone metastases and is suitable for study of interactions between tumor and bone cells and evaluation of new therapeutic modalities.

Ann Biomed Eng, 2003 Oct, 31(9), 1077 - 83
Characteristics of blood flow resistance under transverse vibration: red blood cell suspension in Dextran-40; Shin S et al.; Vibration under shear flow causes the reduction of flow resistance for shear-thinning fluids . The present study investigates the effect of vibration on the flow resistance of a nonaggregating red blood cell (RBC) suspension with a newly designed pressure-scanning capillary viscometer (PSCV) . The PSCV was originally designed to measure non-Newtonian viscosity continuously over a range of shear rates at a time, which was slightly modified and used for the present study . Low-frequency vibration was applied perpendicular to the direction of the flow . The effect of the transverse vibration was investigated for both Newtonian fluids and nonaggregating RBC suspensions . The experimental results showed that the vibration had no effect on the flow resistance of the Newtonian fluids . However, the vibration caused a reduction of the flow resistance of the RBC suspension . The reduction of the flow resistance was strongly dependent on both frequency and amplitude of vibration.

Cryobiology, 2003 Oct, 47(2), 109 - 24
Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents; Thirumala S et al.; In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells . Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO) . Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(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 canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg){cpa}=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp){cpa}=46.4-56.0kJ/mol (11.1-13.4kcal/mol) . These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice . The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.

J Pharmacol Sci, 2003 Oct, 93(2), 210 - 3
Effect of volatile anesthetics on steroidogenesis in isolated bovine adrenocortical fasciculata cells; Masaki E et al.; To examine effects of volatile anesthetics (VAs) on steroidogenesis, cell suspensions of isolated bovine adrenocortical cells were incubated with several steroidogenic agents in the presence or absence of halothane and sevoflurane . The adrenocortical cells were dispersed by trypsin digestion of bovine adrenal cortex . The cortisol level was measured fluorometrically . VAs inhibited adrenocorticotropic hormone-, acetylcholine-, angiotensin-II-, and KCl-stimulated steroidogenesis in a concentration-dependent manner with extracellular Ca(2+) . However, dibutyryl cyclic adenosine monophosphate-stimulated steroidogenesis was not inhibited by VAs . These results suggest that VAs inhibit steroidogenesis by blocking Ca(2+)-influx from the extracellular space without influencing the action of intracellular cyclic nucleotides.

Comp Biochem Physiol A, 1985, 82(4), 915 - 24
The Bohr effect of the blood in rainbow trout (Salmo gairdnerii) . A comparative study with human blood, using precise oxygen equilibrium curves and the Adair model; Vorger P; 1 . The Bohr effects of trout blood (which exhibits the Root effect) and of human blood were compared . Precise oxygen equilibria were measured with an automatic recording system, on normal trout red blood cell suspensions at pH 7.6 - 8.6, at 10 and 20 degrees C, and on normal human red blood cell suspensions at pH 6.8 - 8.0, at 37 degrees C . 2 . The data were fitted to the Adair's stepwise oxygenation model which describes experimental curves with four constants ki (i = 1-4) . 3 . Adair's scheme successfully fits the equilibrium data for trout and human blood, in the range of conditions examined . 4 . The R-state Bohr effect (d log k4/ d pH), is very large in trout blood, indicating a large pH dependence of the R structure, as opposed to human blood . 5 . The T-state Bohr effect (d log k1/ d pH), and the overall Bohr effect (d log Pm/ d pH), are equivalent in trout and human blood . 6 . The overall Bohr effect is essentially accounted for by the first and fourth oxygenation steps in trout blood and shows a significant effect of temperature . 7 . The data attribute a major role to Hb4 in trout blood isotherms and confirm the importance of the C-termini of Beta chains in Bohr and Root effects.

Przegl Lek, 2003, 60 Suppl 5, 5 - 8
{The problems of immunological diagnosis of childhood acute leukemia and non-Hodgkin's lymphoma}; Pituch-Noworolska A; The immunophenotyping of leukaemia and non-Hodgkin's lymphoma cells is based on staining the cells with monoclonal antibodies against surface and cytoplasmic determinants followed with flow cytometry analysis . The problems of immuno-phenotyping are associated with technical difficulties, changes in expression of determinants and the rare types of leukaemia and haematological disorders typical for newborns and infants . The lack of blast cells within cell suspension obtained for test may be the result of bone marrow disorder (aplastic anaemia, preleukaemic cytopenia) or technical pitfall . The changed expression of determinants on blastic cells observed as weak expression or overexpression or atypical combination of determinants requires a careful interpretation . In the diagnosis of rare types of acute leukaemia (e.g . erythroleukaemia, megakaryoblastic leukaemia, mixed lineage or undifferentiated leukaemia) the additional monoclonal antibodies beyond routine set are needed . A special concern is necessary in diagnosis of newborns and infants leukaemia or bone marrow disorders like myelodisplastic syndrome particularly in children with other systemic diseases e.g . congenital immunological deficiencies, Down's syndrome . The problems of immunophenotyping in non-Hodgkin's lymphoma are frequently associated with obtaining a representative material e.g . surgical tumour biopsy, lymph node . In some case the differential diagnosis including small round cell tumours and anaplastic type of lymphoma is necessary what requires an additional set of monoclonal antibodies . Despite of modern technology, morphology, immunophenotyping and histopathology remain the standard of complex diagnosis of lymphoproliferative diseases and haematopoietic disorders in children.

Shi Yan Sheng Wu Xue Bao, 2003 Aug, 36(4), 275 - 8
{Improved camptothecin production by cell lines of Camptotheca acuminata}; Liu WZ; The concentration of camptothecin was determined in different tissues of Camptotheca acuminata seedling . The concentrations of camptothecin in new leaves and roots were significantly higher than in other tissues . However, the concentration of camptothecin declined with leaves becoming old . The induction of callus and cell suspension cultures from younger leaves of Camptotheca acuminata was observed . Cell lines were selected with improved camptothecin production as 0.02%.

Biotechnol Bioeng, 2003 Dec 5, 84(5), 597 - 610
On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy; Cannizzaro C et al.; In this work, dielectric spectroscopy was used to monitor two CHO perfusion culture experiments (B14 and B16) . The capacitance of the cell suspension was recorded every 20 minutes over an excitation frequency range of 0.2 MHz to 10.0 MHz . A phase plot of the capacitance at a low excitation frequency vs . the value at a higher frequency proved to be an accurate indicator of the major transition points of the culture, i.e., maximum cell viability, end of lactate consumption, point of zero viability . For both experiments, the capacitance signal correlated very well (R(2) >0.98) with viable cell number up to concentrations of 1 x 10(7) cells/mL . Visual observation of the capacitance spectra indicated that changes in the capacitance relative to frequency were related to the cellular morphology . A multivariate model was developed using off-line data that could predict the median cell diameter within a single experiment (B14) with an error of 0.34 microm (2%) . Upon extension to a subsequent experiment (B16), the predicted error was 1.18 microm (9%) .

Anal Chem, 2003 Jul 15, 75(14), 3581 - 6
Microfluidic device for single-cell analysis; Wheeler AR et al.; We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells . The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell . Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements . These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

Aquat Toxicol, 2003 Dec 10, 65(4), 361 - 76
DNA integrity determination in marine invertebrates by Fast Micromethod; Jaksic Z et al.; This study was focused toward the adaptation of the previously developed Fast Micromethod for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment . The Fast Micromethod detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates . The procedure is based on the ability of the specific fluorochrome dye PicoGreen to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations . The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs) . The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml(-1) and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5 . The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M . galloprovincialis gills homogenate . DNA damage and loss of DNA integrity were detected after induction by different doses of (gamma-rays, generated by 137Cs 1800 Ci; 0-500 rad in marine sponge S . domuncula cells up to SSFx(-1) values 0.082 +/- 0.012 for the highest radiation dose) . Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin-Fe(II) complex 0-50 or 0-83 microg ml(-1) (microM)) with native DNA from cotton-spinner H . tubulosa and mussel M . galloprovincialis gills homogenate yielded values of 0.537 +/- 0.072 and 0.130 +/- 0.018, respectively . In vivo experiments with mussel M . galloprovincialis gills homogenate by 4-nitroquinoline-N-oxide (NQO; 0-1 microg g(-1) NQO mussel) and benzo{a}pyrene (B{a}P; 0-20 microg g(-1) B{a}P mussel) indicated SSFx(-1) values of 0.121 +/- 0.016 and 0.090 +/- 0.007, respectively, for the highest applied doses of chemical xenobiotics . The analytical technique described here allows simple and fast analysis of DNA integrity, requires very short time for multiple analyses (less than 3 h) and even less than 100 ng DNA per single well (50 ng DNA isolated from cotton-spinner, 12,500 sponge cells or about 10 mg of mussel gills homogenate) in a microplate . This makes the Fast Micromethod applicable for the measurement of DNA integrity of small samples for genotoxicity assessment (biomonitoring), the effects of genotoxins on lower marine taxa or sessile invertebrates in marine environment (e.g . sponges, mussels) and the estimation of directional changes and harmful effects in the ecosystem.

Mol Genet Genomics, 2003 Nov, 270(3), 253 - 62 Epub 2003 Oct 16.
Analysis of the transcriptional response to Rice Yellow Mottle Virus infection in Oryza sativa indica and japonica cultivars; Ventelon-Debout M et al.; Several cDNA libraries were constructed using mRNA isolated from roots, panicles, cell suspensions and leaves of non-stressed Oryza sativa indica (IR64) and japonica (Azucena) plants, from wounded leaves, and from leaves of both cultivars inoculated with Rice Yellow Mottle Virus (RYMV) . A total of 5549 cleaned expressed sequence tags (ESTs) were generated from these libraries . They were classified into functional categories on the basis of homology, and analyzed for redundancy within each library . The expression profiles represented by each library revealed great differences between indica and japonica backgrounds . EST frequencies during the early stages of RYMV infection indicated that changes in the expression of genes involved in energy metabolism and photosynthesis are differentially accentuated in susceptible and partially resistant cultivars . Mapping of these ESTs revealed that several co-localize with previously described resistance gene analogs and QTLs (quantitative trait loci).

Adv Exp Med Biol, 2003, 530, 689 - 96
Rheologic dissimilarities in female and male blood: potential link to development of cardiovascular diseases; Kameneva MV et al.; Oxygen Delivery Index (ODI) was introduced as the ratio of red blood cell concentration (hematocrit) to blood viscosity . The ODI can be considered an indirect characterization of oxygen transport to organs and tissues . ODI was obtained for 98 healthy donors (47 pre-menopausal women and 51 age-matched men) . In this population ODI levels were found to be significantly lower (p < 0.001) in male blood (7.7 +/- 0.3 vs . 8.4 +/- 0.5 in female blood) . Average ODI obtained for 15 cardiac patients (all males) was found to be significantly lower than that for healthy men . In red blood cell suspensions with the same hematocrit, ODI was found to decrease when plasma viscosity was increased via an increase in protein concentration . Additionally, it was found that ODI measured for samples of blood over a wide hematocrit range, obtained by dilution with autologous plasma, possessed the highest values at the hematocrit levels 30 to 40% . The decreased oxygen transport might contribute to the significantly higher morbidity and mortality from cardiovascular diseases for men compared to pre-menopausal women . ODI may be a useful parameter for evaluation of risk of development of cardiovascular disorders.

Ann Hematol . 2003 Oct 14; {Epub ahead of print}
Delayed hemolytic transfusion reaction due to anti-S antibody in patient with anti-Jk(a) autoantibody and multiple alloantibodies; Guastafierro S et al.; We describe the case of a 60-year-old woman with a delayed hemolytic transfusion reaction (DHTR) . She had a history of an ulcerative colitis, blood transfusion because of rectal bleeding, and surgical removal of descendent and sigmoid colon . At admission, laboratory data showed Hb 6.3 g/dL, reticulocytes 120x10(9)/L, serum total bilirubin 1.2 mg/dL (direct bilirubin: 0.2 mg/dL) . Pretransfusion antibody screening procedures were positive . A monospecific autoanti-Jk(a) and three alloantibodies (anti-c, -E, -K) were identified by immunohematologic studies . The patient received two units of crossmatch compatible concentrated red blood cells . Six days later biochemical serum values showed Hb 6.2 g/dL, LDH 975 I.U./L and total bilirubin 2.95 mg/dL (direct 0.35 mg/dL) . Crossmatches with red cell suspension of transfused blood units and a post-transfusion serum were repeatedly positive . Laboratory tests showed the presence of anti-S alloantobody in the serum and eluate . Moreover, pre-transfusion serum of the patient was retrospectively retested: anti-S was not detected . These data suggested a DHTR . The present case is unusual and interesting because of the association of a rare autoanti-Jk(a), non responsible for anemia, and four alloantibodies of which anti-S involved in a DHTR.

Transplantation, 2003 Oct 15, 76(7), 1123 - 30
Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation: methods and techniques; Arbab AS et al.; BACKGROUND: Superparamagnetic iron oxides (SPIO) are being used to label cells for in vivo monitoring by magnetic resonance imaging (MRI) . The purpose of this study is to present protocols using SPIO and a polycationic transfection agent for magnetic labeling of cells as a basis for cellular MRI . METHODS: Various concentrations of ferumoxides (FE)-poly-l-lysine (PLL) complexes were used to magnetically label cells . Iron incorporation into cells along with cell viability and short- and long-term toxicity were evaluated . RESULTS: Rapidly growing cell suspension and adherent cells were effectively labeled by means of endocytosis into endosomes at low concentrations of FE (25 microg/mL media) and PLL (0.75 microg/mL media) . Hematopoietic stem cells and lymphocytes required higher concentrations of PLL (1.5 microg/mL) in serum-free media during initial FE-PLL complex formation before labeling the cells in culture . Total iron concentration in cells depended on the cell type, concentration of FE-PLL complexes in media, cellular density, and incubation time . Iron concentrations after overnight incubation with given FE at 25 microg/mL media resulted in, for example, T cells being labeled with 1 to 3 pg/cell of intracytoplasmic endosomal iron and 15 to 20 pg/cell of intracytoplasmic iron in mesenchymal stem cells compared with 0.01 to 0.1 pg/cell for unlabeled cells . Protocols developed for this study demonstrated no adverse effect on the cell viability, functional capacity, or toxicity . CONCLUSION: This technique can be used to label cells for in vivo MRI tracking of stem cells and lymphocytes . FE at a concentration of 25 to 50 microg/mL with a ratio of SPIO to PLL of 1:0.03 to 1:0.06 would be sufficient to label cells for cellular MRI.

Burns, 2003 Nov, 29(7), 677 - 85
A comparison of keratinocyte cell sprays with and without fibrin glue; Currie LJ et al.; Fibrin glue is an excellent template for cellular migration and has been shown to be an effective delivery system for cultured autologous keratinocytes . We have investigated whether fibrin glue has any benefit on the percentage of epithelial cover when cultured autologous keratinocytes are sprayed onto a freshly debrided wound bed.Three pigs were used for this study . This provided a total of 18 full thickness, vertically orientated wounds, each 4cm in diameter and isolated in PTFE chambers to prevent re-epithelialisation from the wound margins . Eight wounds were sprayed with cultured autologous keratinocytes suspended in 2ml culture medium and eight wounds were sprayed with cultured autologous keratinocytes suspended in 1ml of the fibrin/aprotinin component of Tisseel fibrin glue (Baxter) mixed with 1ml of culture medium . In the latter group the thrombin component of the fibrin glue kit was applied to the wound bed immediately prior to grafting . The remaining two wounds were used as controls and sprayed with either culture medium or fibrin glue without cells . Epithelial cover was calculated in whole-wound biopsies at 3 weeks using image analysis, histology and immunohistochemistry.The cell suspension in fibrin glue appeared to spread more evenly over the wound surface, with no pooling in the inferior aspect of the wound . However, mean epithelial area at 3 weeks in the fibrin group was 1.6cm(2) per wound compared with 1.8cm(2) for the non-fibrin group, as measured by image analysis of digital photographs . There was no statistically significant difference between the two groups (P=0.802) . This surprising result was confirmed by histological analysis of the wound biopsies, with a good correlation between histological and image analysis data (R=0.967) . There was no observable difference in the quality of the epithelium on histological and immunohistological analysis of either group.

Plant Physiol, 2003 Nov, 133(3), 1306 - 13 Epub 2003 Oct 09.
Characterization of leachianone G 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme for the formation of the lavandulyl group of sophoraflavanone G in Sophora flavescens Ait . cell suspension cultures; Zhao P et al.; Leachianone G (LG) 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait . cultured cells . The enzyme transfers a dimethylallyl group to the 2" position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant . This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8 . It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2'-hydroxy function in LG was indispensable to the activity . The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 microm, respectively . Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation . Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2"-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2'-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum . These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.

Mutat Res, 2003 Oct 7, 540(2), 127 - 40
Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002; Moore MM et al.; The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England . This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation . Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay . For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies . Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable . In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans {Environ . Mol . Mutagen . 40 (2002) 292} . While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data . This evaluation should lead to a consensus global approach for data evaluation in the near future.

Pflugers Arch, 2003 Nov, 447(2), 223 - 30 Epub 2003 Oct 08.
Angiotensin-(1-7) stimulates water transport in rat inner medullary collecting duct: evidence for involvement of vasopressin V2 receptors; Magaldi AJ et al.; The peptide angiotensin-(1-7) {Ang-(1-7)} is known to enhance water transport in rat inner medullary collecting duct (IMCD) . The aim of this study was to determine the mechanism of the Ang-(1-7) effect on osmotic water permeability (Pf) . Pf was measured in the normal rat IMCD perfused in vitro in presence of agonists {Ang-(1-7), arginine vasopressin (AVP) and Ang-(3-8)}, and antagonists of the angiotensin and the vasopressin cascade . Ang-(1-7), but not Ang-(3-8), increased Pf significantly . The effect of Ang-(1-7) on Pf was abolished by its selective antagonist, A-779, added before or after Ang-(1-7) . Prostaglandin E2 and the protein kinase A inhibitor H8 also blocked the Ang-(1-7) effect . Blockade of vasopressin V1 receptors by antagonists did not change the Ang-(1-7) effect, but pre-treatment with a V2 antagonist abolished the effect of Ang-(1-7) on Pf . Similarly, pre-treatment with A-779 inhibited AVP's effect on Pf . Forskolin-stimulated Pf was blocked both by A-779 and by the V2 antagonist . Finally, Ang-(1-7) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-779 and the V2 antagonist . These data provide evidence that Ang-(1-7) interacts via its receptor with the AVP V2 system through a mechanism involving adenylate-cyclase activation.

Planta Med, 2003 Aug, 69(8), 739 - 44
The bioconversion process of deoxypodophyllotoxin with Linum flavum cell cultures; Koulman A et al.; The in vitro cell suspension culture of Linum flavum is able to convert high amounts of the 2,7'-cyclolignan deoxypodophyllotoxin into 6-methoxypodophyllotoxin 7- O-glucoside . We studied this conversion in detail by monitoring the intermediates and side-products after feeding different concentrations of deoxypodophyllotoxin . At a low concentration (0.1 mM) deoxypodophyllotoxin is rapidly converted into 6-methoxypodophyllotoxin 7- O-glucoside, 6-methoxypodophyllotoxin and traces of beta-peltatin and podophyllotoxin . The feeding of 0.5 and 2.0 mM also shows a rapid conversion into 6-methoxypodophyllotoxin 7- O-glucoside, but a delayed formation of 6-methoxypodophyllotoxin and beta-peltatin . By using different extraction methods we delivered proof in favour of the hypothesis that a part of the deoxypodophyllotoxin after uptake is temporarily stored as beta-peltatin glucoside.

Planta Med, 2003 Aug, 69(8), 733 - 8
A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris; Koulman A et al.; In the roots of Anthriscus sylvestris 12 different lignans were detected . Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A . sylvestris . In the cell suspension cultures, which were initiated for this study, trace amounts of deoxypodophyllotoxin could be detected . With these cell suspension cultures we carried out feeding experiments using deoxypodophyllotoxin, yatein and, anhydropodorhizol . Yatein had a toxic effect on the cell cultures and was, like anhydropodorhizol, not converted into any detectable product . Deoxypodophyllotoxin, in contrast, was converted into podophyllotoxin, yielding significantly higher concentration than measured in whole plants.

Gynecol Obstet Invest, 2003, 56(3), 143 - 7 Epub 2003 Sep 29.
The role of nitric oxide on apoptosis in human luteinized granulosa cells . Immunocytochemical evidence; Jee BC et al.; This study was designed to investigate the role of nitric oxide (NO) on the apoptosis of human luteinized granulosa cells and its possible pathways . Granulosa cell suspensions were incubated for 48 h after adding NO donor (S-nitroso-N-acetyl-penicillamine, SNAP) and NO synthase inhibitor (nitro-L-arginine methyl ester, L-NAME) at different concentrations . Apoptosis was examined using a terminal deoxynucleotide-transferase-mediated dUTP-biotin nick end labeling method in 70 patients, and immunocytochemical staining was performed for six apoptosis-related proteins in 50 patients . Apoptotic rates were significantly lower in cells incubated with 0.5 mM SNAP, but higher with 0.5, 1.0, and 5.0 mM L-NAME . SNAP (0.5 mM) lowered the expression of Fas and p53 in luteinized granulosa cells, but Bcl-2 expression was increased, and Fas ligand or Bax remained unchanged . Using L-NAME (0.5 and 5.0 mM), the expression of p53 and Bax was increased, but Bcl-2 was unchanged . Fas/Fas ligands were also activated especially in 5.0 mM L-NAME . In conclusion, NO may inhibit apoptosis via decreased Fas and p53, and increased Bcl-2 expression in human luteinized granulosa cells .

J Anat, 2003 Sep, 203(3), 323 - 8
In vivo time-course of the angiogenic response induced by multiple myeloma plasma cells in the chick embryo chorioallantoic membrane; Ribatti D et al.; In this study, we set out to make a fine characterization of the angiogenic response induced by plasma cells obtained from patients with active-multiple myeloma (MM), in comparison with cells obtained from patients with non-active MM and benign lesions such as monoclonal gammopathy of undetermined significance (MGUS), in the chick embryo chorioallantoic membrane (CAM) assay . To achieve this we investigated the time-course of the angiogenic response induced by gelatin sponges soaked in the cell suspensions and implanted on the CAM surface from day 8 to day 12 of incubation by evaluating the number of vessels, of the vessel bifurcation and the intervascular distance at 24, 48, 72 and 96 h after the implants . The results show that plasma cell suspensions obtained from patients with active MM induce a vasoproliferative response that was significantly higher than that induced by cell suspensions obtained from patients with non-active MM or with MGUS, which is also a function of the day of implantation . In fact, implants made from day 8 to day 10 induce a strong angiogenic response, whereas those made from day 11 to day 12 do not . This finding might depend on the fact that CAM endothelium exhibits an intrinsically high mitotic rate until day 10 . Thereafter, the endothelial mitotic index declines rapidly, and consequently cell suspensions implanted on the CAM of successively older embryos are not able to induce a vasoproliferative response in parallel with the reduced rates of growth of the CAM's endothelial cells.

Int J Food Microbiol, 2003 Nov 15, 88(1), 1 - 9
High sucrose concentration protects E . coli against high pressure inactivation but not against high pressure sensitization to the lactoperoxidase system; Van Opstal I et al.; The inactivation of Escherichia coli by high hydrostatic pressure treatment at up to 550 MPa and 20 degrees C was studied in potassium phosphate buffer containing high concentrations of sucrose . E . coli strain MG1655 was pressure-sensitive in the absence of sucrose, but became highly pressure resistant in the presence of 10% to 50% (w/v) sucrose . The pressure resistance of E . coli strain LMM1010, a previously described derivative of MG1655 that is pressure resistant in the absence of sucrose, was further increased in the presence of sucrose, to a similar level as for strain MG1655 in the presence of sucrose . When cell suspensions of either strain were stored after pressure treatment for 24 h at 20 degrees C, a further reduction of the plate counts indicative of pressure induced sublethal injury was observed, that was positively correlated with pressure intensity and negatively with sucrose concentration . Addition of the lactoperoxidase system to the cell suspensions strongly enhanced high pressure inactivation of E . coli at high sucrose concentrations . Using a pressure intensity of only 250 MPa, both E . coli strains were sensitized for the lactoperoxidase system in up to 30% (w/v) sucrose, resulting in at least 10(6)-fold inactivation within 24 h or less after pressure treatment . For comparison, a pressure treatment at 250 MPa in the absence of the lactoperoxidase system did not cause any inactivation of either strain even in the absence of sucrose . At sucrose concentrations above 30% (w/v), no or very little inactivation occurred even in the presence of the lactoperoxidase system.

ASAIO J, 2003 Sep-Oct, 49(5), 537 - 42
Polyethylene glycol additives reduce hemolysis in red blood cell suspensions exposed to mechanical stress; Kameneva MV et al.; Mechanical damage to blood cells is of considerable concern in the development and use of circulatory assist devices and other blood contacting systems . Furthermore, hemodilution with saline, dextran, and other plasma expanders applied during extracorporeal circulation and dialysis increases red blood cell (RBC) susceptibility to the high shear stresses associated with these procedures . In this paper, we present polyethylene glycol (PEG) as a potential erythrocyte protective agent against mechanically induced cellular trauma . Bovine RBCs were subjected to mechanical stress induced by rolling stainless steel shots through RBC suspensions for a constant exposure time . The suspensions were prepared at a hematocrit of 30% in various media: PEG (20,000 molecular weight), autologous bovine plasma, Dextran 40 solution, and phosphate buffered saline (PBS) . RBC suspensions in Dextran 40 were prepared at a viscosity similar to the PEG suspensions . We found the hemolysis level of RBCs suspended in plasma and in PEG solutions to be several times lower (p < 0.001) than in the Dextran and PBS solutions . No statistically significant difference was found between the hemolysis that occurred in suspensions of RBCs in autologous plasma and in 2.0% PEG solutions . Even PEG concentration as low as 0.1% reduced hemolysis by more than 40% compared with PBS or the same concentration of Dextran in suspension medium . Our data demonstrate the efficacy of PEG molecules in reducing mechanical trauma to erythrocytes and suggest the potential for using PEG in assisted circulation, dialysis, and other procedures where RBCs are subjected to extensive mechanical stress.

Planta, 2004 Jan, 218(3), 456 - 9 Epub 2003 Oct 02.
cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures; Nagashima S et al.; A cDNA encoding UDP-glucose: formononetin 7- O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L . cell-suspension cultures using probes from Scutellaria baicalensis UDP-glucose: flavonoid 7- O-glucosyltransferase . The open reading frame of the UGT73F1 cDNA encodes a 441-amino-acid protein with a predicted molecular mass of 48.7 kDa . The deduced amino acid sequence showed that the protein is related to the stress-inducible glucosyltransferases . UGT73F1 mRNA was not detected in untreated G . echinata cultures but was transiently induced by treatment with yeast extract . Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli and purified to near homogeneity by nickel chelate chromatography . The purified recombinant enzyme was selective for isoflavonoid, formononetin and daidzein as substrates, while flavonoids and various tested non-flavonoid compounds were poor substrates.

Neuro Endocrinol Lett, 2003 Jun-Aug, 24(3-4), 269 - 73
Ultrastructure of pinealocytes in mice implanted with Colon 38 adenocarcinoma; Karasek M et al.; OBJECTIVES: Relationship between the pineal gland and neoplastic disease has been repeatedly shown in many both experimental and clinical studies . However, morphological studies of the pineal gland in animals with experimentally-induced tumors are rare . Therefore, we decided to investigate the ultrastructure of pinealocytes in mice with implanted Colon 38 adenocarcinoma . MATERIAL AND METHODS: Male adult B6D2F1 mice were used in this study . The animals were divided into two groups . Eight mice were subcutaneously implanted with Colon 38 cell suspension, whereas the other eight intact animals served as controls . Three weeks after tumor implantation four animals from each group were sacrificed by spinal cord dislocation at 12:00 h or 24:00 h . The pineal glands were removed and processed for electron microscopic studies . The cross-sectional areas of the pinealocyte and its nucleus, and relative volume of mitochondria, Golgi apparatus, lysosomes, granular endoplasmic reticulum, and lipid droplets, as well as the number of dense-core vesicles were estimated using a digital analyzer connected on-line to IBM-PC computer . Statistical analysis of the data was performed using Student's t-test and Snedecor F test . RESULTS: In the pineal glands of the tumor-bearing animals killed during the daytime diminished size of pinealocytes and their nuclei, decreased relative volume of granular endoplasmic reticulum, lysosomes, and lipid droplets as well as decreased number of dense-core vesicles were observed . On the contrary, the relative volumes of mitochondria and Golgi apparatus were increased in these animals . In the tumor-bearing animals killed at night, however, increased cross-sectional areas of pinealocytes, and decreased number of dense-core vesicles were observed . CONCLUSIONS: The results of our study suggest that the presence of the malignant tumor influences the morphology of pineal cells in mice . Considering the fact that different patterns of ultrastructural changes were demonstrated in pinealocytes in different tumor types in various species, it seems that the character of the ultrastructural changes observed in tumor-bearing animals depends on the animal species and tumor type.

Cancer Res, 2003 Sep 15, 63(18), 5970 - 7
Multicellular resistance to tirapazamine is due to restricted extravascular transport: a pharmacokinetic/pharmacodynamic study in HT29 multicellular layer cultures; Hicks KO et al.; In common with other bioreductive drugs, metabolic reduction is required for activation of the benzotriazine-di-N-oxide tirapazamine (TPZ) in hypoxic regions of tumors . This same metabolism also consumes the drug as it diffuses, impeding its penetration into hypoxic tissue . In this study, we develop a pharmacokinetic (PK)/pharmacodynamic (PD) model for TPZ that explicitly includes its diffusion characteristics as measured in multicellular layer (MCL) cultures of HT29 colon carcinoma cells . The kinetics of TPZ metabolism to its mono-N-oxide derivative SR 4317, determined by high-performance liquid chromatography using anoxic HT29 single cell suspensions, demonstrated both a first order and saturable (K(m) = 3.6 micro M) component . Cell killing, assessed by clonogenic assay under the same conditions, demonstrated an approximately quadratic concentration dependence and linear time dependence . TPZ transport through MCLs, determined under hyperoxic conditions (95% O(2)) to suppress reductive metabolism, provided a concentration-independent diffusion coefficient of 0.40 x 10(-6) cm(2)s(-1) . Under anoxia, this transport was strongly suppressed and was well predicted by the single cell metabolism parameters (scaled to the cell density in MCLs) . These PK (transport) and PD (cytotoxicity) parameters were used to calculate cell killing as a function of distance in anoxic HT29 MCLs after the addition of TPZ to both sides of the MCL . The predicted average cell kill was in good agreement with measured values, which showed much less killing than for single cell suspensions under the same conditions . The success of this PK/PD model in predicting response in MCL shows that inefficient transport, rather than changes in intrinsic sensitivity, is responsible for TPZ resistance in these three-dimensional cell cultures and suggests that optimization of transport properties is a high priority in developing second-generation TPZ analogues.

J Clin Virol, 2003 Dec, 28(3), 317 - 22
A simplified cytomegalovirus pp65 antigenemia assay procedure; Gratacap-Cavallier B et al.; A simplified cytomegalovirus (CMV) pp65 antigenemia assay using a one-step erythrocyte lysis, fixation and permeabilization process was compared with a standard protocol, the CMV CINAkit (Argene Biosoft) assay . The results were comparable, both quantitatively and qualitatively . The new method saves time . It also provides flexibility because the cell suspension can be stored so that test completion can be deferred if so desired.

Acta Neurochir Suppl, 2003, 87, 169 - 74
Neural stem/progenitor cells survive and differentiate better in PD rats than in normal rats; Sun ZH et al.; To investigate the effects of grafted neural stem/mesencephalic progenitor cells (NSCs/MP) on rotational behavior of Parkinson's disease (PD) rats and the influence of intracerebral environment on NSCs/MP, we observed the survival and differentiation of NSCs/MP transplanted into 6-hydroxydopamine (6-OHDA)-lesioned and intact striatums . NSCs/MP were prepared from E(11-15) rats and proliferated in serum-free medium with bFGF for several weeks . One day after being primed with serum/dbcAMP to differentiate, cell suspensions were grafted into 6-OHDA-lesioned and intact striatums respectively . It had been found that NSCs/MP were able to survive better and differentiate into more tyrosine hydroxylase (TH)-positive neurons in 6-OHDA-lesioned striatums than in intact ones, and apomorphine-induced rotations were obviously attenuated in MP graft models . The data suggested that NSCs/MP tend to survive and differentiate into TH-positive neurons in 6-OHDA-lesioned striatums . The data demonstrated that striatums in which DAergic terminals are destroyed by 6-OHDA undergo some changes and thus provide more appropriate conditions for NSCs/MP to differentiate into mature DAergic neurons . Furthermore, the finding that MP had greater relieving effects on rotational behavior than NSCs suggests that NSCs could not be used in clinical therapy of PD unless being induced into MP in vitro before transplantation.

J Microsc, 2003 Oct, 212(Pt 1), 13 - 20
Direct attachment of cell suspensions to high-pressure freezing specimen planchettes; Sawaguchi A et al.; We describe a procedure for high-pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate . Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly-l-lysine-coated planchettes . This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells . Furthermore, the yield of well-frozen cells approaches 100% for every cell type we have tried so far . In this report, we show well-preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands . Immunogold labelling of H+/K+-ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin . The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24-28 h on the planchettes retain their responsiveness to stimulation with histamine.

Biotechnol Lett, 2003 Aug, 25(16), 1375 - 81
Biotransformation of {ring-U-14C}4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-phase system; Schmidt B et al.; The biotransformation of {14C}4-n-nonylphenol (5 mg l(-1); 10 mg l(-1)) by Agrostemma githago cell suspensions was studied using a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) . The highly lipophilic 4-n-nonylphenol was applied via n-hexadecane phase . After 7 d of incubation, more than 85% of applied 4-n-nonylphenol was absorbed by the cells, and 40% was transformed to 10 side-chain monohydroxylated metabolites (two with additional double bond at side-chain) . The primary metabolites were analyzed by GC-EIMS . In the cells, the monohydroxylated products and residual 4-n-nonylphenol were present as glycosides . The method proved to be suitable for the production of primary metabolites of 4-n-nonylphenol on a larger scale for identification purposes and for metabolic profiling of the compound.

Biotechnol Lett, 2003 Sep, 25(17), 1437 - 9
Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola; Lu CT et al.; When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l(-1) to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g(-1) dry wt, 9 mg g(-1) dry wt and 57 mg g(-1) dry wt, respectively . The final biomass (1.3 mg dry wt ml(-1)) was not affected.

Am J Respir Cell Mol Biol, 2004 Apr, 30(4), 491 - 9 Epub 2003 Sep 25.
Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny; Abe S et al.; Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung . Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells . Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients . Whole lung cell suspensions, lung side population (SP) cells, and CD45(+/-) lung cells obtained from male transgenic enhanced green fluorescent protein-expressing mice were transplanted intravenously to total body irradiated female mice . Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1) . Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells . Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice . Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45(+) . CD45(+) cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

Am J Physiol Heart Circ Physiol, 2004 Jan, 286(1), H222 - 9 Epub 2003 Sep 25.
Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation; Baskurt OK et al.; The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model . RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68) . Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period . Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system . Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation . Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related . Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions . These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.

Br J Dermatol, 2003 Sep, 149(3), 506 - 12
A multiparameter flow cytometric analysis of the effect of bexarotene on the epidermis of the psoriatic lesion; Franssen ME et al.; BACKGROUND: A new retinoid, bexarotene (Targretin), was recently investigated in a large multicentre trial for its efficacy and safety in psoriasis . Bexarotene is a novel retinoid X receptor (RXR)-selective ligand . OBJECTIVES: The aim was to study the effect of bexarotene in psoriasis by analysing markers for epidermal differentiation, proliferation and inflammation in epidermal single cell suspensions from lesions of patients with psoriasis treated with various doses of bexarotene . METHODS: Thirty-four patients with moderate to severe plaque psoriasis participated in this study and were assigned in sequence to four different dose regimens: 0.5, 1, 2 and 3 mg kg-1 once daily . Before and after 12 weeks of bexarotene treatment, punch biopsies were taken from lesional skin from which epidermal single cell suspensions were prepared using an optimized thermolysin protocol . A sum of scores was determined for each biopsy site, based on a four-point scale for erythema, induration and desquamation . An improved multiparameter flow cytometric assay was used that enabled simultaneous assessment of epidermal proliferation, various aspects of differentiation and epidermal inflammation . The following variables were measured simultaneously: relative DNA content, relative cell size, keratin (K) 10, K6 and vimentin expression . RESULTS: The psoriasis area and severity index (PASI) and sum of scores for the individual psoriatic lesion each showed a statistically significant decrease of 28% after 12 weeks of bexarotene treatment (P < 0.001) . However, no significant dose-response effect was found . The total percentage of K10+ cells showed a significant increase of 43% (P < 0.01) . The total population of K6 expressing cells did not show significant changes . Regarding the subpopulations of K6 single, K10 single and K6 and 10 co-expressing cells, a significant increase of 77% was seen in the K10+ K6- cells (P < 0.05), a significant decrease of 33% in K10- K6+ cells (P < 0.01), and no significant changes in the remaining population of K10+ K6+ cells . After 12 weeks of treatment with bexarotene no significant changes in epidermal proliferation and inflammation were shown . CONCLUSIONS: The present study indicates a direct effect of RXR activation by bexarotene on the transition of proliferation-associated keratinization into normal keratinization . Although no direct effect of bexarotene on DNA content in the total K10- cells was shown, further studies on subpopulations within the germinative layer such as stem cells and transit amplifying cells might be worthwhile.

J Chromatogr A, 2003 Sep 12, 1012(1), 1 - 10
Modeling of the whole expanded-bed protein adsorption process with yeast cell suspensions as feedstock; Chen WD et al.; Expanded bed adsorption of bovine serum albumin (BSA) directly from a feedstock containing whole yeast cells has been investigated with an anion-exchanger DEAE Spherodex M . In the presence of 6% (w/w) yeast cells, the axial liquid-phase dispersion coefficient was found in the order of 10(-6) m2/s, which felled into the common range of 1.0 x 10(-6)-1.0 x 10(-5) m2/s observed previously without the use of cell suspensions as mobile phase . We found that the static and dynamic binding capacity of BSA decreased with increasing the yeast cell concentration due to the competitive adsorption of cells onto the outer surface of the anion-exchanger . However, because of the small size of the adsorbent, the large pore diffusivity of protein and the favorable column efficiency (low axial dispersion coefficient), the dynamic binding capacity of BSA in the presence of 6% (w/w) cells in the expanded bed reached 86% that of the equilibrium adsorption density . Then, the whole expanded bed adsorption process of BSA in the presence of cells, including feedstock loading, washing and elution steps, was predicted using a mathematical model with parameters all determined independently . In the elution stage, the steric mass-action adsorption isotherm with salt concentration as one of the model parameters was used to predict the step-gradient elution process with salt concentration increases . Computer simulations showed that the model was in good agreement with the experimental results for the whole operation process.

J Inorg Biochem, 2003 Sep 15, 97(1), 69 - 78
Changes in some characteristics between the wild and Al-tolerant coffee (Coffea arabica L.) cell line; Martinez-Estevez M et al.; An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH . LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%) . Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake) . Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.

J Cell Biochem, 2003 Oct 1, 90(2), 287 - 93
Direct, rapid effects of 25-hydroxyvitamin D3 on isolated intestinal cells; Phadnis R et al.; Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D(3) {25(OH)D(3)} has biological activity . In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D(3) resulted in enhanced transport of (45)Ca within 2 min relative to the vehicle controls . We then tested the effect of a range of 25(OH)D(3) concentrations on (45)Ca handling by isolated intestinal cells in time course studies . Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D(3), or the vehicle ethanol (0.01%, final concentration) . Both 25 and 100 nM 25(OH)D(3) resulted in a significant (P < 0.05) reduction in (45)Ca levels, relative to controls, between 1-10 min after treatment, while 300 nM 25(OH)D(3) resulted in a significant increase in (45)Ca levels, relative to controls, after 10 min of incubation . The effect of 100 nM 25(OH)D(3) (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25-dihydroxyvitamin D(3) . In cell preparations from 14- or 28-week old birds 100nM 25(OH)D(3) had no effect, relative to vehicle controls . Incubation of cells with 2 microM BAY K8644, a calcium channel activator, stimulated (45)Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 microM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls) . Finally, cells were treated with 100 nM 25(OH)D(3) or vehicle and samples taken at various times for analyses of protein kinase C and A activities . No effect of 25(OH)D(3) on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D(3) addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions . We conclude that 25(OH)D(3) has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway .

Chirurg, 2003 Sep, 74(9), 802 - 7
{Skin tissue engineering}; Bannasch H et al.; Cultivated epithelial autografts as multilayered, thin sheets represent a common standard in clinically applied tissue engineering substitutes, outnumbering all experimental alternatives . However, the unsatisfying short- and long-term results concerning mechanical stability and scarring require alternatives . The cultivation and transplantation of cultured autologous keratinocytes as a single cell suspension in a fibrin matrix, combined with allogenic skin grafting, has been investigated extensively in athymic nude mice . Wounds can be reliably reepithelialized after a cultivation period of only 14 days . Moreover, the successful combination of keratinocyte fibrin suspension and acellular dermis in an attempt to regenerate full thickness skin defects in a pig model has been demonstrated . The usefulness of subconfluently cultured keratinocytes-which can be harvested very early and are easy to handle-is enhanced by cotransplantation with decellularized dermis.

J Virol Methods, 2003 Oct, 113(1), 1 - 12
Rapid in vivo isolation of gene expression elements using an HSV amplicon system; Huang CY et al.; Short-lived gene expression elements (GEE) represent currently a significant obstacle for gene therapy . To identify GEE such as promoters, enhancers, locus control regions, or insulators, useful for long-term or tissue-specific gene therapy, we developed a GEE trapping strategy in which any sequence can be screened for activity in vivo and the expressing clones can be rapidly isolated . Test sequences are introduced into a herpesvirus (HSV) amplicon vector that expresses green fluorescent protein (GFP) only if the insert has GEE function . The plasmid amplicons can be packaged and used to transduce either cultured cells or any tissue or organ in vivo . Single cell suspensions can then be prepared and GFP positive cells isolated by FACS . After sorting, the plasmid amplicons can be isolated and reintroduced into bacteria, cloning the GEE for further characterization . The CMV promoter was used to demonstrate the utility of the system . The amplicon vector was packaged into herpesvirus virions and transduced into Vero cells, confirming the vector can be packaged . After injection into rat eyes, the packaged amplicon virions were capable of transducing cells and the GFP expressing plasmid amplicons were recovered from rat eye tissues by single cell isolation followed by FACS . This novel amplicon system should prove valuable in identifying and characterizing GEE for use in gene therapy.

J Comp Physiol {B}, 2003 Nov, 173(8), 661 - 7 Epub 2003 Sep 09.
Calcium channels are present in the apical plasma membranes of the hepatopancreatic B-cells of Marsupenaeus japonicus; Zilli L et al.; This study demonstrates the existence of calcium channels in the apical membranes of the hepatopancreatic blister (B) cells of Marsupenaeus japonicus . Using brush-border membrane vesicles we demonstrated that the channel-mediated calcium passive flux was saturable and was stimulated by a transmembrane electrical potential difference and inhibited by barium . We raised a monoclonal antibody (Mab 24A4) against the calcium channel, which allowed us to inhibit the channel-mediated calcium uptake . By immunocytochemistry, using Mab 24A4, we demonstrated that these channels are located at the apical membrane of hepatopancreatic B cells . Finally, by measuring the calcium uptake in R- and B-enriched cell suspensions, we showed that only the plasma membrane of the B cells expresses a channel-mediated calcium uptake inhibited by barium, verapamil and the monoclonal antibody 24A4 . The plasma membrane of R cells did not show calcium channels.

Tissue Eng, 2003 Aug, 9(4), 757 - 66
Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability, morphology, and function; Dvir-Ginzberg M et al.; Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver . Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering . The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method . Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL) . Cell density in scaffolds affected hepatocyte viability as judged by MTT assay . At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids . In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture . Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.

Plant Physiol, 2003 Oct, 133(2), 538 - 48 Epub 2003 Aug 21.
Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea; Woo HH et al.; Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1) . Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development . The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence . Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants . PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division . This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root . Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA . Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots . Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid . Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia . Inhibition of endogenous ethylene did not correct this early senescence phenotype.

J Investig Allergol Clin Immunol, 2003, 13(2), 103 - 7
Low density neutrophils in patients with juvenile idiopathic arthritis; Ronchezel MV et al.; OBJECTIVES: (1) To study the correlation among conventional clinical and laboratory parameters and the relation between the number of lymphocytes and neutrophils (L/N) in cell suspensions from peripheral blood of patients with juvenile idiopathic arthritis (JIA) . (2) To evaluate the L/N relation of JIA patients after an 8 year follow-up period . METHODS: Fifty-one JIA patients (25 female, disease course: 19 systemic, 15 polyarticular, 17 pauciarticular) were enrolled in the study . To measure the L/N relation, we used Boyum's method: The leucocyte separation was done by centrifugation of peripheral blood on Ficoll-Hypaque (FH) gradient, and the number of lymphocytes, monocytes, and neutrophils in 500 cells was determined . The following clinical and laboratory parameters were evaluated: disease activity, number of active and limited joints, functional capacity, erythrocyte sedimentation rate (ESR), and C reactive protein (CRP) . Twenty-four healthy children were used as controls . We also studied 13/51 patients from our Pediatric Rheumatology Unit who had been evaluated by the same method 8 years before . RESULTS: We observed the lowest L/N relation in patients with active disease, especially those with polyarticular course . A statistical correlation was also observed with the acute-phase reactants (ESR and CRP, p < 0.05) . The majority of patients who had presented a low L/N relation at the first evaluation (8 years before) had a worse outcome . CONCLUSION: The measure of L/N relation from peripheral blood could be used as an auxiliary tool in the assessment of the activity and outcome of JIA patients, especially at disease onset.

Stem Cells, 2003, 21(5), 588 - 97
Reconstruction of cartilage, bone, and hematopoietic microenvironment with demineralized bone matrix and bone marrow cells; Gurevitch O et al.; Highly specialized hard tissues, such as cartilage, bone, and stromal microenvironment supporting hematopoiesis, originate from a common type of mesenchymal progenitor cell (MPC) . We hypothesized that MPCs present in bone marrow cell suspension and demineralized bone matrix (DBM) that possess natural conductive and inductive features might constitute a unit containing all the essential elements for purposive bone and cartilage induction . Using a rodent preclinical model, we found that implantation of a composite comprising DBM and MPCs into A) a damaged area of a joint; B) an ablated bone marrow cavity, and C) a calvarial defect resulted in the generation of A) a new osteochondral complex comprising articular cartilage and subchondral bone; B) trabecular bone and stromal microenvironment supporting hematopoiesis, and C) flat bone, respectively . The new tissue formation followed differentiation pathways controlled by site-specific physiological conditions, thus developing tissues that precisely met local demands.

Am J Physiol Renal Physiol, 2004 Jan, 286(1), F170 - 9 Epub 2003 Sep 09.
Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins; Hoffert JD et al.; In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with matrix-assisted laser desorption-ionization-time of flight mass spectrometry . Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly purified IMCD cell fraction in which aquaporin-2 was enriched 10-fold . When DIGE was initially applied to rat inner medullas fractionated into IMCD cells (labeled with Cy3) and non-IMCD cells (labeled with Cy5), we identified 50 highly abundant proteins expressed in the IMCD cells . These proteins, identifiable without subcellular fractionation, included chiefly enzymes, structural proteins, and signaling intermediates . An additional 35 proteins were found predominantly in the non-IMCD cell types . Proteins that were highly enriched in the IMCD fraction included cytokeratin 8, cytokeratin 18, transglutaminase II, aminopeptidase B, T-plastin, heat shock protein (HSP) 27, HSP70, and lactate dehydrogenase A . Semiquantitative immunoblotting and immunohistochemistry confirmed relative expression levels and distribution of selected proteins . An additional 40 IMCD proteins were identified in separate experiments aimed at further enrichment of proteins through optimization of sample loading . These studies document the applicability of a standardized approach to purification of IMCD cells for proteomic analysis of IMCD proteins and demonstrate the feasibility of large scale identification of proteins in the native IMCD cell.

Anal Quant Cytol Histol, 2003 Aug, 25(4), 235 - 42
DNA ploidy as a prognostic factor in rhabdomyosarcoma . Analysis of 35 cases with image cytometry; Pohar-Marinsek Z et al.; OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies . STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS . Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry . DNA ploidy was assessed on the basis of DNA index values . We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival . RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age . A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS . The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS . Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors . We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not . The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms . CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal . Larger, cooperative studies could give statistically more reliable results.

Am J Physiol Regul Integr Comp Physiol, 2003 Oct, 285(4), R873 - 9
Photoperiod modulates the effects of norepinephrine on lymphocyte proliferation in Siberian hamsters; Demas GE et al.; Siberian hamsters (Phodopus sungorus) rely on photoperiod to coordinate seasonally appropriate changes in physiology, including immune function . Immunity is regulated, in part, by the sympathetic nervous system (SNS), although the precise role of the SNS in regulating photoperiodic changes in immunity remains unspecified . The goal of the present study was to examine the contributions of norepinephrine (NE), the predominant neurotransmitter of the SNS, to photoperiodic changes in lymphocyte proliferation . In experiment 1, animals were maintained in long {16:8-h light-dark cycle (16:8 LD)} or short days (8:16 LD) for 10 wk, and splenic NE content was determined . In experiment 2, in vitro splenocyte proliferation in response to mitogenic stimulation (concanavalin A) was assessed in spleen cell suspensions taken from long- or short-day hamsters in which varying concentrations of NE were added to the cultures . In experiment 3, splenocyte proliferation was examined in the presence of NE and selective alpha- and beta-noradrenergic receptor antagonists (phenoxybenzamine and propranolol, respectively) in vitro . Short-day animals had increased splenic NE content compared with long-day animals . Long-day animals had higher proliferation compared with short-day animals independent of NE . NE (1 microM) further suppressed splenocyte proliferation in short but not long days . Last, NE-induced suppression of proliferation in short-day hamsters was blocked by propranolol but not phenoxybenzamine . The present results suggest that NE plays a role in photoperiodic changes in lymphocyte proliferation . Additionally, the data suggest that the effects of NE on proliferation are specific to activation of beta-adrenergic receptors located on splenic tissue . Collectively, these results provide further support that photoperiodic changes in immunity are influenced by changes in SNS activity.

J Comp Physiol {B}, 2003 Nov, 173(8), 679 - 86 Epub 2003 Aug 29.
Differential expression of Na+/D-glucose cotransport in isolated cells of Marsupenaeus japonicus hepatopancreas; Vilella S et al.; D-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated . We have studied Na(+)-dependent D-glucose transport (Na(+)/D-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions . As assessed by brush-border membrane vesicle studies, Na(+)/D-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential . Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic . When assayed in purified R and B cell suspensions, Na(+)/D-glucose cotransport activity was restricted to B cells only . Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na(+)/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions . Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA . The molecular nature of this Na(+)/D-glucose cotransport system is still to be established.

J Neurosci Methods, 2003 Oct 15, 129(1), 49 - 59
The use of flow cytometry to assess neutrophil infiltration in the injured murine spinal cord; Tjoa T et al.; Inflammatory cells, including neutrophils, are likely candidates in promoting early cell death after spinal cord injury . We describe a simple and reliable method for obtaining neutrophils from the injured murine spinal cord for flow cytometric quantification . Mice were subjected to either a moderate or severe spinal cord contusion injury and euthanized 24 h later . The area of maximal damage, designated the epicenter, was prepared for assessment of myeloperoxidase (MPO) activity, quantitative immunocytochemistry, or quantification of immunolabeled neutrophils by flow cytometry . For flow cytometry, a cell suspension was prepared from the epicenter by gentle mechanical disruption . After centrifugation, the pellet was resuspended, immunolabeled for neutrophils, and analyzed . There was no detectable MPO activity in the injured spinal cord . In contrast, neutrophil infiltration was confirmed by immunocytochemistry and found to be significantly greater in the more severely injured group . Flow cytometry, using a standard neutrophil marker, revealed a similar significant increase in immunolabeled cells in the more severely injured group . However, when cell viability was determined in the neutrophil labeled population, no significant difference in the numbers of live neutrophils were noted between the two injured groups . Together, these findings demonstrate an effective method for the detection and quantification of viable neutrophils in the injured murine spinal cord.

J Pathol, 2003 Sep, 201(1), 99 - 108
Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer . A pilot study; Gazi E et al.; Fourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason-graded malignant prostate tissue . Paraffin-embedded tissue samples were analysed by FTIR, after mounting onto a BaF(2) plate and subsequent removal of wax using Citroclear followed by acetone . Cell lines were analysed as aliquots of cell suspension held between two BaF(2) plates . It was found that the ratio of peak areas at 1030 and 1080 cm(-1), corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells . The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen . Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason-graded CaP spectra . Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites .

Ai Zheng, 2003 Jun, 22(6), 607 - 11
{Correlation between the PTEN/MMAC1/TEP1 expression and cell proliferation and apoptosis in human renal cell carcinoma (RCC)}; Li JY et al.; BACKGROUND & OBJECTIVE: PTEN is a tumor suppressor gene with phosphatase activity . It had been proved the aberrant expression and mutation of PTEN in diverse types of human cancer; PTEN is closely associated with occurrence and development of malignant tumor . This study was conducted to investigate the expression of PTEN/MMAC1/TEP1 gene product in human renal cell carcinoma (RCC) and the correlation between the expression of PTEN and the biological behaviors of RCC . METHODS: The PTEN protein of 44 cases of RCC tissues confirmed by pathology after operation, 15 cases of adjacent normal renal tissues, and 10 cases of non-tumor normal renal tissues were assessed using immunohistochemical technique (SP) . Fifteen RCC tissues and 10 normal renal tissues selected respectively from the renal tissues whose PTEN protein expression were positive as well as those from the negative were made to be the cell suspension mingled with paraffin . The proliferative index and the apoptosis incidence were examined by flow cytometry and the correlation between PTEN protein and the proliferation and apoptosis were analyzed . RESULTS: The expression of PTEN protein was mostly located in the renal cell plasma . The positive incidence of expression PTEN protein in RCC was 36.3% which was prominently lower than those in the adjacent normal tissues (77.3%) and the normal tissues (100.00%) (P< 0.01) . There was no significant difference between the different sorts of tissues (P >0.05) . The expression of PTEN in stage I, II is much higher than that in stage III, IV(P< 0.05) . The proliferative index of RCC whose expression of PTEN protein was positive (5.6+/-0.8)% was significantly lower than that of negative (15.6+/-1.6)% (P< 0.01) . While the apoptosis incidence was (6.5+/-1.9)%,which was much higher than that of RCC whose expression was (2.9+/-1.6)% (P< 0.01) . CONCLUSION: The positive expression incidence of PTEN protein significantly decreases in RCC tissues . PTEN protein suppresses carcinoma by inducing the cell cycle to be blocked up in G1 phase and increasing the apoptosis incidence . The assessment of the expression PTEN protein is one of the important indexes of the development and prognosis of RCC.

Ultrasound Med Biol, 2003 Aug, 29(8), 1211 - 22
Bioeffects caused by changes in acoustic cavitation bubble density and cell concentration: a unified explanation based on cell-to-bubble ratio and blast radius; Guzman HR et al.; Acoustic cavitation has been shown to load drugs, proteins and DNA into viable cells as a complex function of acoustic and nonacoustic parameters . To better understand and quantify this functionality, DU145 prostate cancer cell suspensions at different cell concentrations (2.5 x 10(5) to 4.0 x 10(7) cells/mL) were exposed to 500 kHz ultrasound (US) over a range of acoustic energy exposures (2 to 817 J/cm(2); peak negative pressures of 0.64 to 2.96 MPa; exposure times of 120 to 2000 ms) in the presence of different initial concentrations of Optison contrast agent bubbles (3.6 x 10(4) to 9.3 x 10(7) bubbles/mL) . As determined by flow cytometry, molecular uptake of calcein and cell viability both increased with increasing cell density; viability decreased and uptake was unaffected by increasing initial contrast agent concentration . When normalized relative to the initial contrast agent concentration (e.g., cells killed per bubble), bioeffects increased with increasing cell density and decreased with increasing bubble concentration . These varying effects of contrast agent concentration and cell density were unified through an overall correlation with cell-to-bubble ratio . Additional analysis led to estimation of "blast radii" over which bubbles killed or permeabilized cells; these radii were as much as 3 to 90 times the bubble radius . Combined, these results suggest that extensive molecular uptake into cells at high viability occurs for low-energy exposure US applied at a high cell-to-bubble ratio.

Phytochemistry, 2003 Sep, 64(2), 485 - 92
Response of scab-susceptible (McIntosh) and scab-resistant (Liberty) apple tissues to treatment with yeast extract and Venturia inaequalis; Hrazdina G et al.; Yeast extract and Venturia inaequalis treated intact scab-susceptible (McIntosh) and scab-resistant (Liberty) apple plants and their organs were analyzed for phenolic metabolites . The major phenolic compounds found in both non-treated and treated leaves were phloridzin and phloretin which accumulated in mM concentrations . Untreated and treated stems and roots contained only phloridzin as the major detectable metabolite during the course of the investigation . The accumulation of phloridzin and phloretin was not developmentally regulated, since they were present in both young and old leaves, and also in the intercellular washings of both scab-susceptible and scab-resistant plants . The major metabolites of both McIntosh and Liberty fruits were cinnamyl glucose and p-coumarylquinic acid, which increased 20-fold in Liberty fruit upon yeast extract treatment . The same compounds increased only 2-fold in McIntosh fruits . Minor compounds in the fruits of both cultivars were p-coumaric acid, phloridzin and phloretin, the latter compound being present at the threshold of detection . Biphenyl and dibenzofuran compounds, the major metabolites of elicitor treated Liberty cell suspension cultures, could not be detected in the intact plants . These results indicate differential response of plant organs and cell suspension cultures to elicitor treatment or pathogen invasion.

Phytochemistry, 2003 Sep, 64(2), 453 - 8
Beta-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum; Kranz K et al.; S-Adenosyl-L-methionine:beta-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L . (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin . The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of beta-peltatin in position 6 thus forming beta-peltatin-A methylether . This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin . The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 degrees C . The enzyme activity is strongly inhibited by MnSO(4), FeCl(3), FeSO(4) and ZnSO(4) as well as S-adenosyl-homocysteine . Mg(2+) and EDTA did not influence the methylation of beta-peltatin . Substrate saturation curves were obtained for S-adenosyl-methionine and beta-peltatin and apparent K(m)-values of 15 microM and 40 microM, respectively, were determined for these substrates . Substrate inhibition was observed for beta-peltatin . No other lignan substrate tested nor caffeic acid were accepted . The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:beta-peltatin 6-O-methyltransferase . Highest specific activities of beta-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.

Phytochemistry, 2003 Sep, 64(2), 445 - 52
Functional expression of cinnamate 4-hydroxylase from Ammi majus L; Hubner S et al.; Total RNA was isolated from dark-grown cell suspension cultures of Ammi majus L . that had been induced with fungal elicitor or treated with water for control and used as template with cytochrome P450-specific primers for DD-RT-PCR amplifications . A cDNA clone was generated from the elicited transcripts and assigned to cinnamate 4-monooxygenase based on sequence alignments and functional expression in yeast cells . Comparison of the translated polypeptide with database accessions of heterologous cytochrome P450 monooxygenases revealed a high degree of similarity (99.6%) with 98.6% identity to cinnamic acid 4-hydroxylase from parsley, documenting the close evolutionary relationship within the Apiaceae family . Maximal activity of the Ammi hydroxylase in yeast microsomes was determined at 25 degrees C and in the pH range of 6.5-7.0 reaching 2.5 pkat/mg on average . An apparent K(m) of 8.9 microM was determined for cinnamate . Preincubations with psoralen or 8-methoxypsoralen added up to 100 microM in the presence or absence of NADPH hardly affected the turnover rate . A . majus cell cultures accumulate sets of O-prenylated umbelliferones and linear furanocoumarins besides lignin-like compounds upon treatment with elicitor, and cinnamic acid 4-hydroxylase catalyzes the initial reaction leading from the general into the various phenylpropanoid branch pathways . Correspondingly, the hydroxylase transcript abundance was induced in the elicited cells.

Morfologiia, 2003, 123(3), 17 - 20
{Development of human brain neural/progenitor cells after transplantation into the brain of adult rats}; Aleksandrova MA et al.; The purpose of the present study was the investigation of human neural stem/progenitor cells (SPC) cultured in vitro, with special reference to their capacity for grafting, migration and differentiation after transplantation into adult rat brain . SPC were isolated from the brain of 9-week-old human embryos and were cultured in a selective medium for 3 weeks . For transplantation, cell suspension or whole neurospheres were used; they were studied 4 weeks following the transplantation in hippocampus, striatum and lateral ventricle of adult rat brain . For the analysis of transplanted SPC, various histological and immunohistochemical staining methods were applied (bisbenzidine, BrdU, antibodies against human nuclei, vimentin, beta-tubulin, neurofilaments, GFAP), that allowed an independent evaluation of their state and differentiation . Transplanted human brain SPC were shown to survive well for one month in all the areas of adult rat brain without immunosuppression . Cells from suspension transplants actively migrated and differentiated into neurons and glial cells . Meanwhile, cell migration from the transplanted whole neurospheres was limited or absent due to the formation of glial barrier.

Magn Reson Med, 2003 Sep, 50(3), 515 - 21
Isotropic susceptibility shift under MAS: the origin of the split water resonances in 1H MAS NMR spectra of cell suspensions; Chen JH et al.; Bulk susceptibility variations in a multiphase system such as cultured cells and tissue have two manifestations: a dipolar field component outside the regular heterogenous region which introduces linebroadening, and an isotropic field part which results in a frequency shift . Previous NMR studies have emphasized the utility of magic angle spinning for averaging the dipolar component, particularly if the spins of interest are limited to one phase of a multiphase system such as a sample of liquid with air pockets or glass beads . However, in analyzing spectra from complex multiphase systems, such as cell suspensions and tissues, etc., the isotropic part is often neglected, leading to questionable interpretation of experimental results . The present study demonstrates that under magic angle spinning, the water resonance in NMR experiments of cell suspensions is split into two resolved peaks due to the isotropic susceptibility shift . These two peaks are assigned to a central core of cell free water and an outer cylindrical ring of tightly packed cells in close association with water . A comprehensive theory for this splitting is provided based on a coaxis cylinder model with different susceptibilities . The frequency difference is shown to be dependent on the susceptibility difference and also on the angle of the rotor in the magnetic field . The splitting distance of the two water peaks can be used to measure the susceptibility difference of water in these two phases . The susceptibility difference was measured for three different cell types: 3T3 F442A preadipocyte cells, mouse embryonic stem cells, and human red blood cells .

Z Naturforsch {C}, 2003 Jul-Aug, 58(7-8), 605 - 8
Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells; Lan WZ et al.; In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs . compared with the control, respectively . The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor . Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs . non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.

Haematologica, 2003 Aug, 88(8), 864 - 73
Expression of CD10 by B-chronic lymphocytic leukemia cells undergoing apoptosis in vivo and in vitro; Morabito F et al.; BACKGROUND AND OBJECTIVES: B-cell chronic lymphocytic leukemia (B-CLL) is an accumulating disease of slowly proliferating cells . CD10 is not normally expressed on the surface of B-CLL cells . The aim of this study was to ascertain whether B-CLL cells, induced into apoptosis, expressed surface CD10, since a correlation between apoptosis and CD10 expression has been demonstrated . DESIGN AND METHODS: Peripheral blood cells from 31 untreated B-CLL patients were induced into apoptosis by etoposide, fludarabine or Ga(mu)-Ab treatment and tested for CD10 expression by flow cytometry . Normal CD5+ B cells were also induced into apoptosis and tested for CD10 expression . RESULTS: CD10 positive cells were absent in B-CLL cell suspensions, but were detected following in vitro culture, and their appearance paralleled that of apoptotic cells . Treatment with etoposide, fludarabine or Ga(mu)-Ab enhanced both apoptosis and CD10 expression . Inhibition of apoptosis by VAD-fmk or Ga(delta)-Ab prevented CD10 expression . Cell separation tests following induction of apoptosis demonstrated that CD10+ cells were apoptotic . CD10+ cells were observed in the peripheral blood of two patients within a few hours following fludarabine infusion . In another patient, who failed to respond, no CD10+ cells were seen . Expression of CD10 was observed also in normal CD5+ B cells when these were induced into apoptosis . INTERPRETATION AND CONCLUSIONS: This study demonstrates that B-CLL cells, as well as normal CD5+ B cells, become CD10+ following apoptosis induction in vitro . Some of the data obtained also suggest a use for CD10 to monitor apoptosis of B-CLL in a clinical setting.

Haematologica, 2003 Aug, 88(8), 845 - 52
Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential; in 't Anker PS et al.; BACKGROUND AND OBJECTIVES: We previously found that human fetal lung is a rich source of mesenchymal stem cells (MSC) . Here we characterize and analyze the frequency and function of MSC in other second-trimester fetal tissues . DESIGN AND METHODS: Single cell suspensions of fetal bone marrow (BM), liver, lung, and spleen were made and analyzed by flow cytometry for the expression of CD90, CD105, CD166, SH3, SH4, HLA-ABC, HLA-DR, CD34 and CD45 . We assessed the frequency of MSC by limiting dilution assay . RESULTS: The frequency of MSC in BM was significantly higher than in liver, lung, and spleen (p<0.05) . On primary non-expanded cells from fetal liver, lung and spleen the number of cells positive for mesenchymal markers was significantly higher within the CD34 positive population than within the CD34 negative population . The phenotype of the culture-expanded MSC was similar for all fetal tissues, i.e . CD90, CD105, CD166, SH3, SH4 and HLA-ABC positive and CD34, CD45 and HLA-DR negative . Culture-expanded cells from all tissues were able to differentiate along adipogenic and osteogenic pathways . However, adipogenic differentiation was less in MSC derived from spleen, and osteogenic differentiation was reduced in liver-derived MSC (p<0.05) . INTERPRETATION AND CONCLUSIONS: Our results indicate that culture-expanded MSC derived from second-trimester fetal tissues, although phenotypically similar, exhibit heterogeneity in differentiating potential . We speculate that these differences may be relevant for the clinical application of MSC.

Lipids, 2003 Jun, 38(6), 651 - 6
Properties of lysophosphatidylcholine acyltransferase from Brassica napus cultures; Furukawa-Stoffer TL et al.; Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA . LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition . LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L . cv Jet Neuf) was assayed using {1-14C}acyl-CoA as the fatty acyl donor . LPCAT activity was optimal at neutral pH and 35 degrees C, and was inhibited by 50% at a BSA concentration of 3 mg mL(-1) . At acyl-CoA concentrations above 20 microM, LPCAT activity was more specific for oleoyl (18:1)-CoA than stearoyl (18:0)- and palmitoyl (16:0)-CoA . Lauroyl (12:0)-CoA, however, was not an effective acyl donor . LPC species containing 12:0, 16:0, 18:0, or 18:1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12:0-LPC was somewhat less effective as a substrate at lower concentrations . The failure of LPCAT to catalyze the incorporation of a 12:0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B . napus expressing a medium-chain thioesterase.

Biol Reprod, 2003 Dec, 69(6), 1940 - 4 Epub 2003 Aug 20.
Germ cell transplantation in an azoospermic Klinefelter bull; Joerg H et al.; Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes . A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization . In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient . The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture . A pulsatile administration of GnRH was performed to stimulate spermatogenesis . The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient . Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo . After slaughtering, meiotic preparations were performed . The injected germ cells did not undergo spermatogenesis . Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis . The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact . Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis . Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients . Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.

Exp Dermatol, 2003 Aug, 12(4), 510 - 3
Photoperiod modulates melanoma growth in C57BL/6 mice; Lang R et al.; Seasonal variations can be found in almost any parameter of an organism's biochemistry, physiology, endocrinology, and behaviour . This phenomenon, generally called photoperiodism, results from one of the major functions of the circadian system, i.e . the translation of environmental information into rhythmic intraorganismic signals, which then regulate or influence physiology and pathology . We induced melanoma in three groups of syngeneic C57BL/6 mice synchronised to different photoperiods (8, 12, or 18 h of light within 24-h days) by subcutaneous injections of HFH18 melanoma cell suspensions . All animals from all three photoperiodic groups developed exponentially growing tumors . The average tumor volume on day 31 post injection was significantly smaller in animals exposed to light/dark conditions (LD) 8 : 16 h as compared with animals held in LD 18 : 6 h and intermediate in animals from the equinox group . These results indicate that C57BL/6 mice react to photoperiod, which can exert a significant effect on tumor growth.

Bioorg Med Chem, 2003 Sep 1, 11(18), 3913 - 9
In vivo monitoring of alkaloid metabolism in hybrid plant cell cultures by 2D cryo-NMR without labelling; Hinse C et al.; Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth . ex Kurz and Rhazya stricta Decaisne were carried out . When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13C . Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800) . Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe that the alkaloid enters two metabolic routes . Vinorine is intracellularly transformed on route I through vellosimine and 10-deoxysarpagine into sarpagine . On route II, the alkaloid is converted by hydroxylation through vomilenine into the glucoside raucaffricine . Intracellular alkaloid concentrations of approximately 500 microM are measurable in vivo with cryogenic NMR technology.

Vaccine, 2003 Sep 8, 21(25-26), 4011 - 21
Structural characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies; Smith ML et al.; Several subunit vaccine antigens have been successfully expressed in plants and recently the hepatitis B surface antigen (HBsAg), expressed in potatoes, was shown to be orally immunogenic in animal studies . However, to date, a detailed analysis of the plant-derived antigen is lacking . Herein, we comprehensively characterize the structure and post-translational processing of HBsAg from potato tuber and two plant cell suspension cultures . The HBsAg was found to accumulate intracellularly as tubular structures, with a complex size distribution, differing substantially from the virus-like particle (VLP) preparations of the current commercial vaccines . Extensive disulfide-bond cross-linking, which is important for immunogenicity, was evident and 21-37% of total HBsAg protein displayed epitopes which correlate with vaccine potency . The significance of these results with regard to the production of cost-effective orally delivered vaccines is discussed.

Mol Imaging, 2002 Apr-Jun, 1(2), 102 - 7
Annexin V-CLIO: a nanoparticle for detecting apoptosis by MRI; Schellenberger EA et al.; Annexin V, which recognizes the phosphatidylserine of apoptotic cells, was conjugated to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI) . The resulting nanoparticle had an average of 2.7 annexin V proteins linked per CLIO nanoparticle through disulfide bonds . Using camptothecin to induce apoptosis, a mixture of Jurkat T cells (69% healthy and 31% apoptotic) was incubated with annexin V-CLIO and was applied to magnetic columns . The result was an almost complete removal of the apoptotic cells (> 99%) . In a phantom MRI experiment, untreated control cells (12% apoptotic cells, 88% healthy cells) and camptothecin-treated cells (65% apoptotic cells, 35% healthy cells) were incubated with either annexin V-CLIO (1.0, 0.5, and 0.1 microgram Fe/mL) or with unlabeled CLIO . A significant signal decrease of camptothecin-treated cells relative to untreated cells was observed even at the lowest concentration tested . Unmodified CLIO failed to cause a significant signal change of apoptotic cells . Hence, annexin V-CLIO allowed the identification of cell suspensions containing apoptotic cells by MRI even at very low concentrations of magnetic substrate . Conjugation of annexin V to CLIO affords a strategy for the development of a MRI imaging probe for detecting apoptosis.

Acta Cytol, 2003 Jul-Aug, 47(4), 616 - 23
Immunocytochemical detection of p16INK4a protein in scraped cervical cells; Pientong C et al.; OBJECTIVE: To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening . STUDY DESIGN: We took duplicate cervical scrapes from each participant, the first for a Pap smear and the second for p16INK4a protein detection . From a 50-microL cell suspension prepared from the scrape rinsing, a 10-microL aliquot was dropped in a 5-mm-diameter circle on a glass slide, air dried and fixed in 0.1% formal saline (1 hour) and in 95% ethanol (10 minutes) . Using the immunocytochemical technique, slides from 30 samples of each Pap diagnosis class were stained sequentially with mouse monoclonal anti-p16INK4a (primary antibody), biotinylated goat antimouse IgG (secondary antibody), horse-radish peroxidase-labelled streptavidin and 3,3'-diaminobenzidine and mixed hydrogen peroxide, then counterstained with hematoxylin . A positive sample had to contain > or = 3 immunoreactive cells . Results were confirmed by western blot analysis of lysates from the remaining 40 microL of each cervical cell suspension . RESULTS: Samples were grouped as control (normal cervical cells), mild dysplasia (ASCUS, LSIL) and high abnormality (HSIL, SCC) . Using the immunocytochemical technique, > 95% of the positive (SiHa cells) but 0% of the negative controls (human embryonic lung fibroblast cells) showed immunoreactive cells . All slides displayed a clear background without mucus, and positive cells were stained in both the cytoplasm and nucleus . p16INK4a Protein was detected in 17 of 30 (56.67%) ASCUS and 10 of 30 (33.33%) LSIL and increased with the degree of abnormality to 93.33% (28 of 30) and 96.67% (29 of 30) in the HSIL and SCC group, respectively . Normal cervical cells and degenerated malignant cells were nonimmunoreactive . Western blot analysis confirmed similar positive samples in the low-abnormality group, while the whole high-abnormality group was immunoreactive . A sampling error might have caused the 2 HSIL and 1 SCC sample to be negative using our immunocytochemical technique . CONCLUSION: p16INK4a Protein detection in scraped cervical cells using the immunocytochemical technique correlated with western blot analysis and was nontraumatic and precise . It offers a significant diagnostic adjunct to the Pap test for cervical cancer screening.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2003 Jul, 17(4), 282 - 5
{Synapses developing process of neuroblasts after acute spinal cord transplantation in rats}; Xue Y et al.; OBJECTIVE: To explore the potential possibility of synaptic connection and 3D adhesion between fetal spinal cord cell suspension (FSCS) and host, and to observe the synapses developing process of FSCS transplantation . METHODS: Spinal cord injury model produced in 42 Wistar rats on T7 by use of modified Allen's impact method (10 g x 5 cm); 3 days after injury, 20 microliters FSCS with a density of 1 x 10(5)/microliter prepared from E14 rat were injected into the epicenter of the traumatized cavity . Animals were sacrificed after 2, 4, 6, 8, 10 and 12 weeks of transplantation, the graft survival, its differentiation and integration with the host were observed by light and electronmicroscopic study as well as immunohistochemical assay (NF, GFAP, CGRP, 5-HT) . RESULTS: In the transplantation area, the neuroblasts stretched out the terminal endings 4 weeks after implantation, followed by the presenting of the pre- and postsynaptic membrane . After 8 weeks, the dense or developed projections were observed in the pre- and postsynaptic membrane; the synaptic cleft filled with the high electron dense substance . All the spherical clear vesicles, granular vesicles, elliptical vesicles and flattened-f type vesicles were seen under the electronmicroscope . After 10 weeks, the axosomatic, dendrosomatic, dendro-dendritic, axo-axonic, dendro-axonic synapses coexisted . Light microscopy showed that the graft cell grew gradually . Immunohistochemical assay showed that NF, 5-HT, CGRP and GFAP positive fibers were in the graft . Synapses, gliafibers and blood brain barrier integrated each other . CONCLUSION: (1) The transplanted FSCS can develop mature synapses with miscellaneous synaptic vesicles in the acute injured spinal cord, host injury cavity wall may induce the FSCS into 3D adhesion . (2) Co-existence of different type of synapse and the immunohistochemistry findings indicate the possibility of synaptic connection between FSCS and host.

Pediatr Radiol, 2003 Oct, 33(10), 688 - 92 Epub 2003 Aug 08.
Pinhole imaging of 131I-metaiodobenzylguanidine (131I-MIBG) in an animal model of neuroblastoma; Accorsi R et al.; PURPOSE: To evaluate (131)I-MIBG scintigraphic localization of xenotransplanted and spontaneously arising neuroblastomas in murine models of high-risk neuroblastoma . METHODS: Neuroblastoma xenografts were created by inoculation of human neuroblastoma cell suspensions into the subcutaneous flanks of athymic nude mice . In addition, spontaneous paraspinal neuroblastomas were detected by direct palpation in MYCN transgenic mice . After measured tumor volumes exceeded 200 mm(3), each mouse received an intraperitoneal injection of 18 muCi/g (131)I-metaiodobenzylguanidine ((131)I-MIBG) . Pinhole scintigraphy was performed to evaluate the MIBG biodistribution and to attempt to visualize the tumors . Each mouse was imaged on a gamma camera equipped with a 3-mm pinhole on one head and an HEGP collimator on the other . RESULTS: Images demonstrated absorption of radiolabeled MIBG and visualization of tumors . Analysis of the images allowed for quantification of relative MIBG uptake and for determination of linear and area measurements of the tumors . CONCLUSION: High-energy pinhole imaging effectively demonstrates uptake of radiolabeled MIBG by human neuroblastoma tumors in murine laboratory models . This technique allows for in vivo assessment of tumor burden . In the future, we plan to use this method to evaluate sensitivity for detecting metastatic spread as well as investigating the therapeutic efficacy of high-dose (131)I-MIBG in combination with radiosensitizing agents.

Cryo Letters, 2003 May-Jun, 24(3), 161 - 70
A numerical study of cell behaviour in a ternary solution during the freezing process; Luo D et al.; Using a continuum model for multi-component phase change system, the freezing of cell suspension in a ternary solution, H2O-NaCl-CPA (cryoprotective agent) inside a flat bag is investigated numerically in this study . The temperature and phase change history, intracellular water loss, and the volume change of the cells at different locations inside cell suspension are calculated . Numerical results reveal that although the sample boundary is cooled at a constant rate, different locations inside the sample experienced different temperature changes and cooling rates . The highest cooling rates occur at internal locations . The cell volume change is location-dependent.

Kisaengchunghak Chapchi, 1983 Dec, 21(2), 234 - 240
{Changes In The Pathogenicity Of Naegleria Fowleri By Several Brain Passage In Mice}; Lee DK et al.; The pathogenicity of free-living amoeba, Naegleria fowleri, is influenced according to the strain, cultural condition and host (Culbertson et al., 1968; Carter, 1970; Wong et al., 1975) . Phillips (1973) demonstrated that Entamoeba histolytica became avirulent after more than 2 year maintenance in axenic culture in vitro . This study was carried out to compare the difference in pathogenicity between two strains of N . fowleri, one of a prolonged maintenance in axenic medium and the other one obtained by serial brain passage in mice . The 0 strain was that N . fowleri had cultivated axenically more than 7 years in CGVS medium . The 2-1 strain was obtained from the brain of mouse inoculated intranasally with a strain, which was from the mouse brain infected with 0 strain, and cultured for 15 weeks until the beginning of this experiment . White male mice weighing 18-22 g were used . Mice were anesthetized by an intraperitoneal injection of about 1 mg secobarbital, and inoculated intranasally with 10 x 10(4) live N . fowleri trophozoites in a 5 microliter cell suspension . Sluggish behaviour, nervousness, rotation and leg paralysis were developed earlier and more frequently in the 2-1 experimental group than the control 0 group . Pathological changes such as inflammatory and necrotic lesion were observed in the olfactory and anterior portion of brain, and these changes were more extensive in the 2-1 group . The edematous and inflammatory changes in lung were demonstrated in mice died after 13th day post-inoculation . The experimental mice of 2-1 group began to die suddenly from 7th day post-inoculation, and the survival time in 2-1 group mice was shorter than 0 group mice . The typical primary amoebic meningoencephalitis was developed in the mice inoculated intranasally with N . fowleri . The prolonged maintenance of N . fowleri amoebae in axenic CGVS medium was observed to have lost their original pathogenicity for mice, but their pathogenicity was restored by serial brain passage in mice.

Endothelium, 2002, 9(1), 37 - 44
Metabolism of dynorphins by peptidases of pulmonary artery endothelial cells; Zharikova A et al.; Degradation of several dynorphins by peptidases expressed in cultured porcine pulmonary artery endothelial cells was studied by incubation of the peptide in cell suspensions followed by electrospray ionization and tandem mass spectrometric analyses . Under the in vitro conditions applied, only the metabolism of dynorphin A1-8 occurred in a significant extent . Studies involving specific peptidase inhibitors indicated that mainly bestatin-sensitive aminopeptidases, thiorphan-sensitive endopeptidases, and cFPAAF-pAB-sensitive endopeptidases expressed by the endothelial cells were involved in the process that converted dynorphin A1-8 to dynorphin A2-8, dynorphin A1-6, and leucine enkephalin (dynorphin A1-5), respectively . These peptidases may form a metabolic barrier for the cellular penetration of intact dynorphin A1-8 and/or control effects of the circulating peptide on endothelial opioid receptors of the cells.

FEMS Immunol Med Microbiol, 2003 Aug 18, 38(1), 65 - 70
Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium; Shrivastava R et al.; The cells of the immune system form a strong line of defence against foreign substances . The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro . 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air . At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated . Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages . The reduction by splenic and peritoneal macrophages was similar . Total thymocytes reduced 54% of the Cr (VI) . Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated . Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells . The reduction in the intestinal loop in situ was 100% . The time taken by each cell type for the peak reduction to Cr (VI) was markedly different . The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium . The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.

Zhonghua Yi Xue Za Zhi, 2003 Jun 10, 83(11), 967 - 71
{Integrin alpha3beta1 mediates hepatocellular carcinoma cell adhesion and chemotaxis to type IV collagen}; Fu BH et al.; OBJECTIVE: To investigate the effects of Integrin alpha(3)beta(1) on the adhesion and chemotaxis of hepatocellular carcinoma (HCC) cells to type IV collagen (Col IV) . METHODS: (1) HCC cells were culture and suspension of HCC cells was made . Anti-alpha(3) and Anti-beta(1) were added into the HCC cell suspension . Flow cytometry was used to determine the expression of integrin alpha(3)beta(1) on the surface of HCC . (2) 5 micro g/ml Col IV was used to coat a cell with the diameter of 25 mm . Digested HCC cells were added . Anti-alpha(3) and Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml respectively were added into the cell suspension . Before and after the addition of Anti-alpha(3) and Anti-beta(1), micropipette technique was used to measure the adhesion force of HCC on Col IV-coated surface, as function of the square of internal radius of micropipette and the critical negative pressure needed to detach a single HCC cell away from the substrate . (3) Col IV of the concentration of 600 micro g/ml was added into the dual micropipettes . Then the dual micropipettes were led towards the HCC cells . A HCC cell was made to seal the openings of the 2 micropipettes with different parts of the cell contacting Col IV in different micropipettes . The pseudopod protrusion was observed dynamically and recorded with tape recorder . The length of pseudopod was measured and plotted against the chemotactic time so as to obtain a pseudopod growth curve . RESULTS: (1) The expression rates of integrin subunit alpha(3) and beta(1) on the surface of HCC cells were 95.55% and 95.78% respectively . (2) The adhesion force of HCC cells to the 5 micro g/ml Col IV-coated surface was 932 +/- 134 (x 10(-10) N, n = 60) . Upon treatment of the HCC cells with Anti-alpha(3) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 42% and 49%, to 536 +/- 122 (x 10(-10) N, n = 60) and 476 +/- 63 (x 10(-10) N, n = 60) respectively . Upon treatment of the HCC cells with Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 52% and 76%, to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 60) respectively . (3) The length of pseudopod increased along with the chemotactic time . The pseudopod length and growth curve were almost identical in the dual micropipettes when they were filled with Col IV . When Anti-alpha(3) or Anti-beta(1) was added into one of the dual micropipettes, the HCC cell pseudopod protrusion was almost blocked completely, while the HCC cell pseudopod in the opposite micropipette became more evident . CONCLUSION: Integrin alpha(3)beta(1) is an important constituent receptor in mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.

J Comp Neurol, 2003 Sep 15, 464(2), 172 - 9
Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease; Marchionini DM et al.; One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue . Unfortunately, up to 95% of grafted cells die, many via apoptosis . In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function . We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions . In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control . Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions . In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl) . However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival . These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo . In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation .

J Allergy Clin Immunol, 2003 Aug, 112(2), 411 - 9
Surface expression of Fc epsilon RI on Langerhans' cells of clinically uninvolved skin is associated with disease activity in atopic dermatitis, allergic asthma, and rhinitis; Semper AE et al.; BACKGROUND: Fc epsilon RI expressed on the surface of human epidermal Langerhans' cells facilitates uptake of IgE-associated allergens and plays a pivotal role in the pathogenesis of atopic dermatitis . Seminal results from studies investigating Langerhans' cell Fc epsilon RI in skin biopsy sections or epidermal cell suspensions demonstrate the highest receptor expression in lesional skin of patients with active atopic dermatitis . OBJECTIVE: We sought to investigate and localize Fc epsilon RI expression on Langerhans' cells within a minimally disturbed tissue environment in clinically uninvolved skin and to compare receptor expression between healthy donors and patients with atopic dermatitis or other allergic diseases . METHODS: Intact epidermal sheets from skin suction blisters, immunofluorescently stained with Langerhans' cell markers and anti-Fc epsilon RI alpha (mAbs 15E5 and 22E7) or anti-IgE, were examined by means of confocal microscopy . Samples incubated with anti-Fc epsilon RI alpha before or after cell fixation-permeabilization were compared to discriminate between cytoplasmic and membrane localization . RESULTS: Cytoplasmic Fc epsilon RI alpha chain was found in Langerhans' cells from all donors, irrespective of atopic status . Surface Fc epsilon RI-bound IgE was detected in the skin of individuals with active atopic dermatitis and in the skin of those with active asthma or rhinitis . No surface Fc epsilon RI was expressed in the skin of patients with a clinical history of atopic dermatitis, asthma, or rhinitis whose disease was in remission or in the skin of nonatopic individuals . CONCLUSION: In clinically uninvolved skin, Langerhans' cell-surface Fc epsilon RI expression is not only linked to atopic dermatitis but is also generally associated with allergic disease . This supports the concept of a systemic regulatory mechanism associated with active allergic disease, which is further aggravated by local inflammation in atopic skin lesions.

Biotechnol Lett, 2003 Jul, 25(13), 1055 - 9
Production of alkaloids by in vitro culture of Erythrina americana Miller; San Miguel-Chavez R et al.; The production of erythroidines and other alkaloids was studied in cotyledons, callus and cell suspension cultures of Erythrina americana Miller . The cell suspension cultures, grown in Murashige & Skoog medium with naphthaleneacetic acid (3 mg l(-1)) and kinetin (2 mg l(-1)), produced 89 and 17 microg alpha- and beta-erythroidines respectively per g dry wt.

Biotechnol Lett, 2003 Jun, 25(11), 835 - 9
Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L . (grapevine) suspension culture; Conn S et al.; Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins . AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V . vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation . An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating . The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx . 28-29%, with no apparent preference for anthocyanin species . This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2 . The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.

Zhonghua Wai Ke Za Zhi, 2003 Mar, 41(3), 214 - 7
{Experiment on fibroblast-PGA complexes cultured in rotary cell culture system}; He C et al.; OBJECTIVE: To investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro . METHODS: Rabbit skin fibroblasts of passage 2 were seeded at 2 x 10(6) cell per cm(3) onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS) . Attachment of cells in foams was observed by cell-counting after trypsin digestion . The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition . Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope . RESULTS: Numbers of cells adhering to polymers increased gradually during an initial period of 24 hours . Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70% + 2.16% vs . 31.50% +/- 3.54%; 56.36% +/- 3.18% vs . 34.28% +/- 3.16%; 66.32% +/- 4.60% vs . 37.38% +/- 4.66%; P < 0.01) . The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks . CONCLUSIONS: RCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.

Biophys J, 2003 Aug, 85(2), 719 - 29
Effective conductivity of a suspension of permeabilized cells: a theoretical analysis; Pavlin M et al.; During the electroporation cell membrane undergoes structural changes, which increase the membrane conductivity and consequently lead to a change in effective conductivity of a cell suspension . To correlate microscopic membrane changes to macroscopic changes in conductivity of a suspension, we analyzed the effective conductivity theoretically, using two different approaches: numerically, using the finite elements method; and analytically, by using the equivalence principle . We derived the equation, which connects membrane conductivity with effective conductivity of the cell suspension . The changes in effective conductivity were analyzed for different parameters: cell volume fraction, membrane and medium conductivity, critical transmembrane potential, and cell orientation . In our analysis we used a tensor form of the effective conductivity, thus taking into account the anisotropic nature of the cell electropermeabilization and rotation of the cells . To determine the effect of cell rotation, as questioned by some authors, the difference between conductivity of a cell suspension with normally distributed orientations and parallel orientation was also calculated, and determined to be <10% . The presented theory provides a theoretical basis for the analysis of measurements of the effective conductivity during electroporation.

Biotechnol Lett, 2003 Jan, 25(1), 47 - 9
Cell death unlikely contributes to taxol production in fungal elicitor-induced cell suspension cultures of Taxus chinensis; Lan WZ et al.; Cell suspension cultures of Taxus chinensis, with 20, 40 and 100 mg fungal elicitor l(-1) from Aspergillus niger, underwent rapid cell death after 24 h, which was about 2, 3.7 and 5-fold of that of the control . At the same time, Taxol production was increased, respectively, to about 5, 8 and 3-fold of that of the control . Inhibition of phenolics biosynthesis resulted in a 150% increase in cell death but a 54% decrease in Taxol production compared with 40 mg elicitor l(-1) alone . O2-free N2 inhibited cell death but had little effect on Taxol production as induced by 40 mg fungal elicitor l(-1).

Anal Quant Cytol Histol, 2003 Jun, 25(3), 139 - 45
Flow and image cytometric DNA ploidy, including 5c exceeding cells, of serous borderline malignant ovarian tumors . Correlation with clinicopathologic characteristics; Flezar MS et al.; OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors . STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors . One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM . RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1 . ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells . There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival . CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells . The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.

Hum Immunol, 2003 Aug, 64(8), 787 - 95
Four-color flow cytometric analysis of peripheral blood donor cell chimerism; Metes D et al.; Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation . Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes . In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples . Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures . By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2% . When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected . Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells . This procedure provided a simple and reliable method in determining early chimerism in transplant recipients . However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.

Bioorg Chem, 2003 Aug, 31(4), 345 - 56
Substrate specificity for the hydroxylation of polyoxygenated 4(20),11-taxadienes by Ginkgo cell suspension cultures; Dai J et al.; Three C-14 oxygenated taxanes isolated from callus cultures of Taxus spp., 2alpha,5alpha,10beta,14beta-tetra-acetoxy-4(20),11-taxadiene 3, 2alpha,5alpha,10beta-triacetoxy-14beta-propionyloxy-4(20),11-taxadiene 4, 2alpha,5alpha,10beta-triacetoxy-14beta-(2-methylbutyryl)-oxy-4(20),11-taxadiene 5, and three deacetylated derivatives of 3, 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene 6, 14beta-hydroxy-2alpha,5alpha,10beta-triacetoxy-4(20),11-taxadiene 7, 10beta,14beta-dihydroxy-2alpha,5alpha-diacetoxy-4(20),11-taxadiene 8, could all be regio- and stereo-selectively hydroxylated at the 9alpha-position by Ginkgo cell suspension cultures to yield a series of new 9alpha,14beta-dihydroxylated taxoids . The effects of functional groups, especially at C-14 of the substrates, on the biotransformation were also investigated . The results revealed that substrates with an acetoxyl group at C-14 could be more efficiently 9alpha-hydroxylated than those with a longer ester chain or a hydroxyl group at C-14 . An acetoxyl or hydroxyl group at C-10 had no effect on the conversion rates of the substrates, but substrates with the hydroxyl group (compared with the acetoxyl analogues) could be converted into 9alpha-hydroxylated products more easily.

Cell Physiol Biochem, 2003, 13(3), 135 - 46
Absence of adhesion triggers differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit adhesiveness and lead to cell death; Walsh MF et al.; BACKGROUND: Upon adhesion, anchorage-dependent cells transmit survival signals from the matrix into the cell . Loss of anchorage leads to anoikis . Resistance to anoikis may influence tumor progression and metastasis . To better understand the pathways that regulate the choice between adhesion and cell death, we examined FAK, c-Src andMAPKinase activities in SW620 human colon cancer cells . METHODS: SW620 cell suspensions were first allowed to adhere to collagen I for 30 minutes and adherent cells were subsequently counted . FAK, p38, c-Src and ERK1/2 phosphorylation were assessed by Western blot in adherent cells and in cells prevented from adhesion by plating unto BSA-pacificated dishes . p38 and FAK were inhibited by SB203580 (20 microM) or by specific FAK antisense nucleotides or FAK siRNA, respectively, and adhesion quantitated . Apoptosis (anoikis) after lack of adhesion was measured colorimetrically in control cells and in cells treated with SB203580 . RESULTS: Adhesion to collagen I nearly doubled FAK phosphorylation at Y397, the autophosphorylation site, and decreased p38 activation by 60% (p<0.001) but did not affect FAK phosphorylation at Y576, the c-Src dependent site . Lowering FAK expression with FAK antisense decreased adhesion to collagen I; the larger decrease in FAK expression obtained with the siRNA (43 +/- 2%) resulted in significantly greater inhibition of adhesion not only to collagen I but also to collagen IV and fibronectin . The p38 inhibitor diminished anoikis and enhanced adhesion . Interestingly, the SB compound also significantly inhibited FAK phosphorylation at Y397 (23 +/- 3% in adherent, 30 +/- 4% in non-adherent cells at 30 minutes and 35 +/- 4% in adherent, 46 +/- 14% in non-adherent cells after 6 hours, p<0.05 for each) and greatly enhanced phosphorylation of ERK1/2, a putative anti-apoptotic component of the MAPK cascade . CONCLUSIONS: In the absence of adhesion, SW620 cells exhibit increased p38 but decreased FAK activation, signals that may promote cell death . Our observations with the p38 inhibitor SB203580 indicate that inside-out signals, from p38 to FAK, may regulate both adhesion and anoikis in SW620 cells . In addition, the data suggest the presence of cross-talk between the pro-apoptotic p38 and anti-apoptotic ERK1/2 pathways .

Exp Biol Med (Maywood), 2003 Jul, 228(7), 850 - 4
Casein binds to the cell membrane and induces intracellular calcium signals in the enteroendocrine cell: a brief communication; Hira T et al.; Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells . However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified . The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1 . The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor . Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring . Intracellular calcium concentrations in STC-1 cells were increased by exposure to alpha-casein or casein sodium, but not to bovine serum albumin . Solubilized STC-1 membranes bound to alpha-casein and casein sodium but did not bind to bovine serum albumin . alpha-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium . Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated . Intracellular calcium responses to alpha-casein were suppressed by an L-type calcium channel blocker . These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.

Z Naturforsch {C}, 2003 May-Jun, 58(5-6), 308 - 12
Sterols and triterpenes in cell culture of Hyssopus officinalis L; Skrzypek Z et al.; Cell suspension cultures from hypocotyl-derived callus of Hyssopus officinalis were found to produce two sterols i . e . beta-sitosterol (1) and stigmasterol (2), as well as several known pentacyclic triterpenes with an oleanene and ursene skeleton . The triterpenes were identified as oleanolic acid (3), ursolic acid (4), 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (5), 2alpha,3beta-dihydroxyurs-12-en-28-oic acid (6), 2alpha,3beta,24-trihydroxyolean-12-en-28-oic acid (7), and 2alpha,3beta,24-trihydroxyurs-12-en-28-oic acid (8) . Compounds 5-8 were isolated as their acetates (6, 8) or bromolactone acetates (5, 7).

Hum Gene Ther, 2003 Jul 1, 14(10), 1017 - 34
Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses; Youil R et al.; First-generation adenovectors have been developed for gene therapy and vaccine applications . The construction of these adenovectors has entailed the use of numerous types of expression cassettes . It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others . This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region . In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII . The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors . Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart . By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions . Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture . This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.

Radiat Med, 2003 May-Jun, 21(3), 120 - 7
Effects of p53 status and wortmannin treatment on potentially lethal damage repair, with emphasis on the response of intratumor quiescent cells; Masunaga S et al.; PURPOSE: To examine the effects of p53 status and wortmannin treatment on potentially lethal damage repair, referring to the response of intratumor quiescent cells . METHODS: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were injected subcutaneously into both hind legs of Balb/cA nude mice . Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors . The mice then received gamma-rays with or without subsequent wortmannin administration . Right after or 24 h after gamma-ray irradiation alone or 24 h after wortmannin administration following irradiation, the tumors were excised, minced, and trypsinized . The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling {quiescent (Q) cells} was determined using immunofluorescence staining for BrdU . The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU . RESULTS: On the whole, larger values of MN frequency and surviving fraction were observed in SAS/mp53 cells than in SAS/neo cells, and Q cells showed lower MN frequencies than total cells . Without wortmannin, SAS/neo tumor cells, especially Q cells within SAS/neo tumors, showed large potentially lethal damage repair (PLDR) capacities, compared with total or Q tumor cells within SAS/mp53 tumors that showed little PLDR capacity . Wortmannin treatment inhibited the PLDR in SAS/neo tumors very effectively, but showed no apparent effect on either total or Q tumor cells within SAS/mp53 tumors . CONCLUSION: PLDR in vivo was thought to be a p53-dependent event whether in total or Q tumor cell populations.

Curr Eye Res, 2003 Jul, 27(1), 25 - 34
RPE cell transplants to non-immune-privileged sites of the eye transform into fibroblast-like cells; Knoernschild T et al.; PURPOSE: In vitreoretinopathy membranes and in vitro, RPE cells lose pigment and transform into fibroblast-like cells . The purpose of this study was to investigate whether the immune privileged status of different eye compartments influence morphological changes of adult RPE cell transplants . METHODS: RPE cells from adult C57BL/6 mice were transplanted either intravitreally (n = 31), subretinally (n = 28), intrachoroidally (n = 21) or subconjunctivally (n = 33) into one eye of BALB/c mice and intravitreally (n = 5) into rd-mice with loss of ACAID (14-16 months) . Eyes were enucleated after 1, 2, 4 and 6 weeks, and transplants investigated by light (n = 128) and electron microscopy (n = 40) . RESULTS: Allogeneic adult RPE cells lost pigment and transformed into spindle shaped cells when injected into the recipient subconjunctiva . In vitreous or subretinal space they remained rounded and pigmented . In the choroid, they were spindle shaped but contained more pigment and were larger than those in the conjunctiva . No inflammatory reaction in the uvea or development of epiretinal membranes was seen . In contrast, in all rd-mice an inflammation of the uvea and development of epiretinal membranes was observed . CONCLUSIONS: Our findings suggest that changes in the environment of RPE in single cell suspension dictate the phenotype the cells adopt . It is possible that changes in microenvironmental factors contribute to the pathogenesis of proliferative vitreoretinal (PVR) membranes.

Int J Cancer, 2003 Sep 20, 106(5), 713 - 22
Neutrophils influence melanoma adhesion and migration under flow conditions; Slattery MJ et al.; We have studied human melanoma cell (C8161) adhesion and migration in response to stimulation by soluble collagen IV (CIV) using a modified Boyden chamber . In this modified chamber, shear flow can be introduced over the cell-substrate interface, affecting tumor cell chemotactic migration through a microporous filter . A relatively high level of intercellular adhesion molecule-1 (ICAM-1) was found on C8161 cells . In contrast, levels of beta(2)-integrins (e.g., LFA-1 and Mac-1), the molecules that would be necessary for C8161 stable adhesion to the endothelium substrate, were found to be very low on these melanoma cells . As a result, C8161 transendothelial migration under a flow condition of 4 dyn/cm(2) decreased by 70% as compared to static migration . When human neutrophils (PMNs) were present in the tumor cell suspension, C8161 migration recovered by 85% over C8161 cells alone under the 4 dyn/cm(2) flow condition . Blocking ICAM-1 on C8161 cells or Mac-1 on PMNs significantly inhibited C8161-PMN adhesion and subsequent C8161 migration through the endothelium under flow conditions . In addition, increased interleukin-8 production and Mac-1 expression by PMNs were detected when they were co-cultured with C8161 melanoma cells . These results suggest that transmigration of C8161 cells under flow conditions can be influenced by PMNs, mediated by Mac-1/ICAM-1 adhesive interactions and enhanced by altered cytokine production .

Biochem J, 2003 Oct 15, 375(Pt 2), 415 - 23
Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms; Price LJ et al.; The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses . In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition . We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use . Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A . myosuroides or from sensitive maize . Purifications of up to 300-fold were necessary to separate the two isoforms . The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity . Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides . However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not . Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

Platelets, 2003 May, 14(3), 131 - 7
Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates; Arnaud F et al.; It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant . This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation . The DMSO solution with or without cell suspension was passed once through the filter . The optimum exchange during unloading of DMSO was determined by varying the flow rates in the external and internal compartments of the HF filter . Initially, buffered solutions of a 5% DMSO solution in the absence of platelets were pumped into the fibers and exchanged against PBS . The residual DMSO was determined by osmometry . The exchange of DMSO across the membrane was flow dependent and also influenced by the chemical nature of the HF fibers . No protocol using a reasonable rate flow through the fibers removed more than 95% of the DMSO in a single pass . The optimum protocol was achieved with polysynthane fibers with an internal flow rate of approximately 20 mi/min and an external flow rate of 100 ml/min . Subsequently, frozen/thawed platelet concentrates in DMSO were washed using centrifugation and compared to the HF filtration method . Platelet quality was assayed by flow cytometry, cell count, morphology and osmotic stress test . Both filtration and centrifugal washing techniques resulted in comparable morphological scores and numbers of discoid cells . When agents reducing platelet activation were added, platelet quality was improved after washing by either technique . The lower platelet osmotic response with HF filtration than with centrifugation while using activation inhibitors was attributed to the remaining amount of the inhibitors . All other parameters tested were similar . The expression of CD62P was equivalent with both techniques, and centrifugation did not activate platelets more than filtration contrary to what was originally anticipated . In conclusion, platelet quality was comparable after washing by either technique but hollow fiber filtration does remove cryoprotectant more rapidly than does centrifugation.

J Allergy Clin Immunol, 2003 Jul, 112(1), 141 - 8
Characterization of dendritic cells from human oral mucosa: a new Langerhans' cell type with high constitutive FcepsilonRI expression; Allam JP et al.; BACKGROUND: The oral mucosa represents a unique immunologic unit with a high frequency of native allergen contact within the gastrointestinal tract in which immune tolerance is the natural outcome of allergen contact . Although Langerhans' cells (LC), known to play a crucial role in initiating allergen-dependent immune responses in the skin, have also been detected in the oral mucosa, little is known about their phenotype and exact physiologic role . OBJECTIVE: To elucidate whether LC from oral mucosa (oLC) differ from skin LC (sLC), these cells were subjected to detailed comparative analysis . METHODS: Crude epidermal and oral mucosa cell suspensions were prepared by trypsinization . oLC and sLC were compared phenotypically by flow cytometry techniques and functionally in T-cell proliferation assays . RESULTS: In contrast to sLC, freshly isolated oLC expressed significantly higher amounts of MHC class I and II, as well as costimulatory molecules CD40, CD80/B7.1, and CD86/B7.2 . oLC displayed FcgammaRIII/CD16 and FcgammaRI/CD64 . Most surprisingly, oLC constitutively expressed the high affinity receptor for IgE (FcepsilonRI) even in nonatopic donors . FcepsilonRI expression on oLC was further increased and correlated with the serum IgE levels in atopic individuals . oLC showed a higher allogeneic stimulatory activity than sLC, whereas the activation of autologous T cells correlated to the FcepsilonRI expression . CONCLUSION: Taken together, our results strongly indicate that oLC profoundly differ from their skin counterparts . The constitutive high expression of FcepsilonRI on oLC could point to particular skills of these cells within the regional immune system of the oral mucosa.

J Endocrinol Invest, 2003 Apr, 26(4), 359 - 63
Development of a meningioma in a patient with acromegaly during octreotide treatment: are there any causal relationships?
De Menis E, Tulipano G, Villa S, Billeci D, Bonfanti C, Pollara P, Pauletto P, Giustina A.
Somatostatin receptors are highly expressed in almost all meningiomas but in this setting their functional role is not clear . A 59-yr-old woman had been treated with octreotide after an unsuccessful operation for a GH-secreting pituitary adenoma . After 8 yr of treatment, a nuclear magnetic resonance (NMR) scan disclosed a 3 cm meningioma of the tentorium . Mean GH was 2.2 ng/ml and IGF-I 325 ng/ml . Meningioma was resected and tissue was digested to obtain tumor cell suspension . Aim of the study was to measure epidermal growth factor (EGF)-induced proliferation of cultured meningioma cells in the presence of either somatostatin or octreotide . Cells were grown to semiconfluency in Dolbecco's modified eagle medium (D-MEM) supplemented with 10% fetal calf serum (FCS) . After 48 h in D-MEM without serum, the medium was replaced by fresh medium plus recombinant EGF (10 ng/ml) and somatostatin or octreotide were added in the final concentrations of 1, 10 and 100 nM . 20 h later 1 microcgCi of 3H-thymidine was added to each well . After 4 h, incorporated radioactivity was measured . While octreotide did not influence significantly cell growth at the three dose tested, somatostatin increased thymidine incorporation dose-dependently (peak 100 nM: 150% +/- 27% vs medium plus EGF, p<0.05) . Octreotide effectively suppressed GH secretion in our acromegalic patient but is unlikely that its long-term use could have stimulated the growth of meningioma since it did not significantly influence the in vitro proliferation of the meningioma cells . These results suggest that somatostatin-mediated proliferative effect on meningioma cells is not mediated by the subtype 2 of the somatostatin receptor.

Proteomics, 2003 Jun, 3(6), 1047 - 59
Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors; Ndimba BK et al.; A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme . The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response . Changes in the cell wall and culture filtrate proteome were profiled . Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) . An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation . The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation . In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase . Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues . The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.

Acta Histochem, 2003, 105(2), 115 - 25
Beta-amyloid peptide-induced blood-brain barrier disruption facilitates T-cell entry into the rat brain; Farkas IG et al.; Activated T-lymphocytes can migrate through the blood-brain barrier (BBB) and are able to invade the central nervous system (CNS) . In the present study, we investigated whether disruption of the BBB leads to enhanced T-cell migration into the CNS . Amyloid-beta peptide 25-35 (A beta) or tumor necrosis factor-alpha (TNFalpha) were administered into the right common carotid artery of adult male Wistar rats . The agents were administered either alone, or were followed by a cell suspension of exogenously activated T-cells . Rats of other groups received activated or non-stimulated T-lymphocytes only . Sagittal brain sections were analyzed with immunohistochemistry of CD3 to reveal the presence of T-lymphocytes within the CNS parenchyma . Administration of activated T-cells alone led to T-cell migration into the brain . Infusion of either substances (A beta or TNFalpha) resulted in T-cell invasion of the CNS even when no exogenous T-cells were added . Infusion of either of the agents together with T-lymphocytes generated a more intense T-lymphocyte migration than in the other groups . Electron microscopic analysis and Evans-blue extravasation studies confirmed parallel disruption of the BBB . Our study demonstrates that A beta and TNFalpha induce enhanced T-lymphocyte migration towards the brain . This effect may be attributed at least partly to dysfunctioning of the BBB, but other mechanisms are also possible.

Int J Radiat Oncol Biol Phys, 2003 Jul 15, 56(4), 1184 - 93
Evaluation of hypoxia-inducible factor-1alpha (HIF-1alpha) as an intrinsic marker of tumor hypoxia in U87 MG human glioblastoma: in vitro and xenograft studies; Vordermark D et al.; PURPOSE: The transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulatory element of the hypoxic response of cells . High protein levels have been linked to poor prognosis in several tumor types, and HIF-1alpha has been suggested as a potential endogenous marker of tumor hypoxia and associated radioresistance . METHODS AND MATERIALS: HIF-1alpha expression following in vitro hypoxia was measured in U87 MG glioblastoma cells by Western blot and flow cytometry . Cell suspensions from U87 MG xenograft tumors grown in SCID mice were assayed by flow cytometry for HIF-1alpha and for pimonidazole as a reference hypoxia marker.After 1 h, 6 h, and 18 h of in vitro hypoxia, a constant increase in HIF-1alpha protein levels with decreasing oxygen concentrations between 20% and <0.02% was observed by both Western blot and flow cytometry, correlating with the pattern of pimonidazole labeling after in vitro hypoxia . In U87 MG xenograft tumors, flow-cytometric analysis of HIF-1alpha and pimonidazole showed a significant correlation of the two markers, but distinction of a HIF-1alpha-positive population was affected by a low dynamic range of the signal . As in published studies for HIF-1alpha and the hypoxic marker EF5, the colocalization of HIF-1alpha and pimonidazole in double-staining experiments was low . CONCLUSIONS: While the in vitro data in U87 MG human glioblastoma cells support the use of HIF-1alpha as an endogenous hypoxia marker, comparison with the standard pimonidazole makes its application to clinical material appear questionable.

Transplant Proc, 2003 Jun, 35(4), 1506 - 7
Removal of CD45+ cells from human fetal pancreas alters immunogenicity in vitro; MacKenzie DA et al.; Human fetal pancreas (HFP) is a potential source of islets for the treatment of diabetes mellitus with the potential for growth and differentiation after transplantation . However, because of the small mass of a given HFP, multiple donors would be required for transplantation, thereby increasing the immunological challenge to the recipient . In this study, we investigate the contribution of hematopoietic cells to the immunogenicity of HFP . Single cell suspensions of HFP were depleted of CD45(+) cells using antibody-conjugated magnetic beads . In vitro mixed lymphocyte islet cultures were established using with CD45-depleted or nondepleted HFP . Depletion of CD45(+) cells resulted in the low levels of IFNgamma production at early time points (day 4), which increased to near normal levels by day 7 . The development of donor-specific CTL was not consistently inhibited by CD45 cell depletion . The data suggests that CD45(+) cells within HFP are capable of stimulating immune responses by the direct pathway of antigen presentation, but that the indirect pathway is also involved in the development of CTL . The inhibition of early IFNgamma release, however, may be beneficial for the survival of transplanted islets . Therefore, the combination of CD45 depletion strategies with standard immunosuppressive drug therapies could result in better long-term survival of transplanted islets.

Zhonghua Yi Xue Za Zhi, 2003 Mar 10, 83(5), 421 - 4
{Effects of chelerythrine on cell proliferation and p27 expression of cardiac fibroblasts modulated by arginine vasopressin}; Zhao LY et al.; OBJECTIVE: To study the effects of chelerythrine, a protein kinase C (PKC) inhibitor, on the cell proliferation and p27 expression of cardiac fibroblasts (CFs) modulated by arginine vasopressin (AVP) and to investigate the intracellular signal transduction mechanisms of AVP in CFs . METHODS: The cultured CFs of neonatal Sprague-Dawley rats were divided into 3 groups: AVP group (10(-7) mol/L AVP was added into the culture), chelerythrine group (10(-6) mol/L chelerythrine and 10(-7) mol/L AVP were added into the culture), and control group . MTT assay was used to evaluate the cell proliferation . The cultured cells were collected and propidium iodide was used to label the DNA so as to identify the cell cycle . Specific mouse-versus-rat p27 protein monoclonal antibody and fluorescein isothiocyanate-labeled secondary antibody were added into the cell suspension to label the p27 protein in the cells . Flow cytometry was used to determine the distribution of cell cycles and p27 expression . RESULTS: (1) . The A value of CFs measured by MTT assay in AVP + chelerythrine group was 0.32 +/- 0.01, significantly lower than that of the AVP group (0.39 +/- 0.01, P < 0.01) . (2) . The percentage of CFs in S stage was 4.4% +/- 1.7% in the AVP + chelerythrine group, lower than those of the AVP group (15.5% +/- 1.4%, P < 0.01) and control group (7.5% +/- 1.0%) . The PI of CFs was 20.9% +/- 1.2% in the AVP + chelerythrine group, significantly lower than that of the AVP group (31.4% +/- 1.5%, P < 0.01) . The PI of the AVP group was significantly lower than that of the control group (26.0% +/- 1.0%, P < 0.01) . The percentage of CFs in G(0)/G(1) stage was 79.1% +/- 1.2% in the AVP + 1 chelerythrine group, significantly higher than that in the AVP group (68.6% +/- 1.5%, P < 0.01) . The percentage of CFs in G(0)/G(1) stage in the AVP group was significantly lower than that in the control group (74.0% +/- 1.0%, P < 0.01) too . (3) . The expression rate of p27 protein was 91.7% +/- 2.2% in the AVP + chelerythrine group, significantly higher than that in the AVP group (63.3% +/- 1.9%, P < 0.01) . CONCLUSION: PKC inhibitor remarkably reverses the CFs proliferation and p27 downregulation induced by AVP . It may be involved in the intracellular signal transduction pathway of AVP in CFs.

Anticancer Res, 2003 Mar-Apr, 23(2B), 1219 - 21
Single-cell irradiation from {211At} astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention; Palm S et al.; New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented . Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation . Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension . The results from each experiment were then fit to a mono-exponential function . From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0).

Anticancer Res, 2003 Mar-Apr, 23(2A), 999 - 1006
Her2/neu analysis in formalin-fixed, paraffin-embedded breast carcinomas: comparison of immunohistochemistry and multiparameter DNA flow cytometry; Leers MP et al.; PURPOSE: The determination of Her2/neu over-expression has become necessary for the selection of breast cancer patients for Herceptin therapy . Immunohistochemistry in combination with FISH is currently the most used method for this detection . The objective of our study was to compare the use of multiparameter DNA flow cytometry (MP-FCM) for this assessment with immunohistochemistry (IHC) . For this purpose the number of positive cells as well as the intensity of the immunofluorescence signal as determined by MP-FCM were compared with IHC scores . MATERIALS AND METHODS: Single cell suspensions from 110 breast cancers were analysed on a DAKO Galaxy flow cytometer after simultaneous (immuno)labeling for cytokeratin, Her2/neu and DNA (propidium iodide) . Next to ploidy and proliferative activity (S-phase fraction), the fraction of Her2/neu-positive epithelial cells was determined . In 25 cases the intensity of the immunofluorescence signal was also determined by MP-FCM in the total cytokeratin+ fraction as well as the cytokeratin+/Her2/neu+fraction . Immunohistochemistry for Her2/neu was done on paraffin sections of all cases using the same polyclonal Her2/neu antibody in a SABC-peroxidase procedure after heat-induced antigen retrieval . RESULTS: Her2/neu overexpression as determined by IHC showed a score of 0 in 30%, 1 in 25%, 2 in 26% and 3 in 19% of the cases . There was a good correlation when comparing the fraction of Her2/neu-positive epithelial cells as determined by MP-FCM and semiquantitative IHC scores (r = 0.89, p < 0.0001) . No correlation was found between the IHC-Scores and the intensity of the Her2/neu- staining in the total epithelial compartment as measured by MP-FCM . However, when restricting the intensity-determination to the Her2/neu-positive epithelial cells, a significant correlation was found between MP-FCM and IHC scores (r = 0.72, p < 0.0001) . CONCLUSION: The results of the current study show that MP-FCM can be a good alternative for the objective quantification of Her2/neu.

Cytometry A, 2003 Jul, 54(1), 66 - 74
Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens; Plander M et al.; BACKGROUND: Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population . Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application . We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis . METHODS: After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested . RESULTS: The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum . Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions . DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide . Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells . CONCLUSIONS: These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies .

Radiology, 2003 Aug, 228(2), 480 - 7 Epub 2003 Jun 20.
Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents; Frank JA et al.; PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents . MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations . Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO . Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment . Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated . RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100% . Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone . For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s . Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles . Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells . CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings . Copyright RSNA, 2003.

Curr Eye Res, 2003 Feb, 26(2), 81 - 8
Autologous transplantation of retinal pigment epithelium after mechanical debridement of Bruch's membrane; Phillips SJ et al.; PURPOSE: To determine whether transplantation of autologous retinal pigment epithelium (RPE) will prevent atrophy of the choriocapillaris and loss of photoreceptors in an area in which the RPE has been mechanically debrided from Bruch's membrane . METHODS: Abrasive debridement of RPE was performed with a metal cannula after localized retinal bleb detachments in two separate areas of the rabbit retina . The RPE cell suspension aspirated from one of the debridement sites was transplanted to the other . The debridement-only site served as control . The transplant and control sites were evaluated after 30 days by color fundus photography, fluorescein angiography, light microscopy and transmission electron microscopy . RESULTS: Compared with debridement only, debridement plus transplantation of RPE resulted in more complete repopulation of the bare Bruch's membrane surface with relative preservation of choriocapillaris and photoreceptors . CONCLUSION: Autologous transplantation of RPE onto an abrasively debrided Bruch's membrane decreases choriocapillaris atrophy and photoreceptor loss.

Rheumatol Int, 2004 Mar, 24(2), 71 - 6 Epub 2003 Jun 17.
Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients; Solomon S et al.; Using single cell suspensions from synovial fluid cells of arthritis patients, we observed differentiation of three-dimensional tissues in vitro . This new model of pannus-like tissue (PLT) might be useful to study pannus tissue formation and differentiation . In the PLT cultures, we observed two cell types, fibroblast-like and macrophage-like cells, defined by their distinct morphology and major histocompatibility complex (MHC) by human leukocyte antigen (HLA) class II expression . We could discriminate several intermediate steps of differentiation which finally led to 3D villi-like structures . Secretion of interferon gamma, interleukin-10, and tumor necrosis factor alpha was measured in the culture supernatants . Using methotrexate at various concentrations, the growth of PLT could be inhibited . We describe definite intermediate steps of differentiation . The present approach could be a suitable model for the in vitro study of pannus tissue formation.

Plant Cell Rep, 2003 Aug, 21(12), 1217 - 20 Epub 2003 Jun 17.
A simple method for enhancing paclitaxel release from Taxus canadensis cell suspension cultures utilizing cell wall digesting enzymes; Roberts SC et al.; Paclitaxel storage in Taxus suspension cell cultures was studied through the simple use of cell wall digesting enzymes . The application of cellulase (1%) and pectolyase (0.1%) to Taxus canadensis suspension cultures induced a significant increase in the paclitaxel present in the extracellular medium while maintaining membrane integrity, suggesting that paclitaxel is stored in the cell wall . The addition of cell wall digesting enzymes to a cell culture bioprocess may be an effective way of enhancing paclitaxel release to the extracellular medium and hence simplify product recovery.

Phytochemistry, 2003 Jul, 63(5), 533 - 41
Use of plant cell cultures to study graminicide effects on lipid metabolism; Price LJ et al.; Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase . Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro . Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants . Acyl lipid biosynthesis in P . annua can be inhibited if sufficiently high concentrations of graminicide are used . The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue . These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing.

J Card Surg, 2003 May-Jun, 18(3), 268 - 73
Clinical cellular cardiomyoplasty: technical considerations; Zhang F et al.; Three patients, all with a history of coronary heart disease, underwent coronary artery bypass grafting and implantation of autologous satellite cells . Satellite cells were isolated from muscle biopsies of the right vastus lateralis muscle after enzymatic treatment . While the heart was still under hypothermic cardioplegia, 4 mL of cell suspension divided into approximately 40 doses was injected into the ventricular wall of the ischemic area . Less than 5 minutes were required to complete the cell implantation . All patients survived the procedure, without obvious arrhythmia, had an uneventful recovery, and were discharged from the hospital . At 3 to 4 months follow-up examination, increased left ventricular ejection fraction, decreased left ventricular diastolic diameter, as well as improved ventricular wall thickness and perfusion at the satellite cell implantation sites were observed . Our experience indicated the safety and early benefit of cellular cardiomyoplasty using autologous satellite cells.

Acta Anaesthesiol Scand, 2003 Jul, 47(6), 702 - 6
Respiratory burst activity of polymorphonuclear cells is dependent on the cell preparation technique; Zhao J et al.; BACKGROUND: Controversial results have been reported regarding the effect of anaesthetics on superoxide anion production during the respiratory burst (RB) of polymorphonuclear cells (PMN) . The differences could be caused by the cell preparation methods and the aim of this study was to compare two techniques . METHODS: RB activity was measured in cell suspensions isolated with the single-step Ficoll procedure and in unfractionated whole blood . Two concentrations of propofol (therapeutic and 10-fold of this, 6 microg ml-1 or 60 microg ml-1) were investigated after cell preparation with both methods . RB was stimulated with Escherichia coli (E . coli), phorbol 12-myristate 13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured by means of fluorescence intensity in a flow cytometer . RESULTS: The percentage of PMNs in whole blood which generate superoxide anions in response to fMLP was significantly lower (2.5 +/- 0.7%; mean +/- SEM) than that in Ficoll isolated cell suspensions (15.1 +/- 1.7%) . Incubation with propofol led to a concentration-related decrease of RB activity in Ficoll separated PMNs after both PMA and fMLP stimulation . No significant effect of propofol was observed on the RB in PMA stimulated whole blood samples . CONCLUSION: The results suggest that the influence of cell preparation methods should be considered when the in vitro effects of anaesthetics on PMN functions are studied with flow cytometric methods.

Virus Genes, 2003, 26(2), 119 - 30
Immunological and regulatory functions of uninfected and virus infected immature and mature subtypes of dendritic cells--a review; Becker Y; In 1868, dendritic cells (DCs) were discovered in human skin by Paul Langerhans using gold staining . These cells were named Langerhans cells (LCs) after their discoverer who, due to their dendrites, regarded them as neurons . One hundred and eleven years were to pass until it was discovered that in vertebrates these cells originate in the bone marrow as monocytes . In the 1980s, DC research was mostly carried out on DCs that are present in different tissues of mice and humans . These studies revealed that after interaction with foreign antigens, skin LCs/DCs migrate through the lymph vessels to the draining lymph nodes and induce the two arms of the immune response . The isolation of DCs from tissue cell suspensions opened the way to studies on the cells' surface proteins and their ability to stimulate immune responses . During the 1990s, studies revealed the role of DCs in the activation of naive T cells in the lymph nodes and the regulatory properties of DCs in lymph nodes, thymus, gut, and spleen . Part A of the review deals with the DC system of human and mice and immunological and regulatory functions of subsets of DCs in the skin with reference to migrating and stationary DCs, as well as the connection between DCs and the nervous system . Furthermore, the origin of both follicular DCs that are present in lymphoid tissues and thymic DCs are discussed . Part B is devoted to virus infections of DCs with an emphasis on infections caused by human herpes viruses . Part C presents the modulation of DC gene expression in response to the influenza virus . Contemporary research focuses on the role of DCs in the immune systems of vertebrates . Moreover, studies are being conducted on the regulatory functions of DCs by tissue cells in different organs of vertebrates.

Int J Cancer, 2003 Aug 20, 106(2), 153 - 9
Spontaneously formed tumorigenic hybrids of Meth A sarcoma cells and macrophages in vivo; Busund LT et al.; We have recently demonstrated that malignant cells can hybridize with tissue macrophages in vitro, giving rise to tumorigenic hybrids . We now demonstrate that this can occur spontaneously in vivo as a result of fusion between inoculated Meth A sarcoma cells and host cells, presumably macrophages . Thus, from tumor cell suspensions prepared by collagenase perfusion and density centrifugation, hybrid cells could be isolated that were neoplastic but in contrast to Meth A expressed macrophage markers and had phagocytic capacity . Their morphologic features were intermediate between Meth A and macrophages . By taking advantage of a semiallogeneic experimental system by inoculation of Meth A cells from BALB/c (H-2 K(d)) into (BALB.K x BALB/c) F(1) (H-2(k/d)), hybrid cells from these tumors could be shown to express MHC antigens of both the Meth A and the host haplotypes . Hybrid cells grew faster than Meth A cells in vivo, indicating acquisition of growth-promoting properties through heterotypic cell fusion .

Ann N Y Acad Sci, 2003 May, 996, 158 - 73
Pluripotent stem cells identified in multiple murine tissues; Howell JC et al.; Pluripotential stem cells (PSCs) have been recently described in many tissues including skeletal muscle, brain, and bone marrow . However, the true nature of these cells is still unclear, and their precise definition has yet to be determined . We hypothesized that a common, rare population of PSCs with a broad tissue differentiation potential can be identified in multiple murine tissues and that these cells are capable of transdifferentiation into cells of different primordial germ layer origins in response to diverse microenvironmental cues . To examine this hypothesis, we isolated phenotypically defined cells from murine skeletal muscle and cultured these cells under different conditions tailored to promote differentiation into several cell types including myocytes . We report here that in conditions permissive for hematopoietic differentiation, muscle-derived CD45(-)Sca-1(+)c-kit(-) cells differentiated into cells expressing hematopoietic-specific mRNA; while in conditions promoting myogenic, neuronal, and adipocytic differentiation, cells morphologically typical of these cell types expressing tissue-specific markers were identified 9-14 days in culture . When CD45(-)Sca-1(+)c-kit(-) cells from muscle or bone marrow were transplanted intracerebellarly into Purkinje cell degenerative (pcd) mice, the behavior of these mice improved 28 days after transplantation relative to mice injected with vehicle alone, suggesting that these cells contributed to the appearance of functional neuronal cells that may have improved the ataxic condition characteristic of these mice . Phenotypic analysis of single cell suspensions prepared from brain, blood, and intestinal epithelium revealed the presence of CD45(-)Sca-1(+)c-kit(-) cells in varying degrees . These studies suggest that a phenotypically common, multipotent cell can be identified in different tissues and that this cell may represent a universal pluripotent stem cell residing at different levels in multiple murine tissues.

Cell Transplant, 2003, 12(3), 235 - 41
Ultrastructural characterization of dissociated embryonic ventral mesencephalic tissue treated with neuroprotectants; Ahn YH et al.; Poor survival and differentiation of grafted dopamine neurons limits the application of clinical transplantation in Parkinson's disease . The survival of grafted dopamine neurons is only improved by a factor of 2-3 by adding neuroprotectants during tissue preparation . We used dye exclusion cell viability and electron microscopy to investigate the effects of the caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural changes in dissociated embryonic mesencephalic cells . In addition, we examined whether the neuroprotectants selectively counteracted specific signs of neurodegeneration . Cell viability decreased significantly over time in both control and treated cell suspensions, but the number of viable cells remaining was significantly higher in tirilazad mesylate-treated cell suspensions . In control samples, the proportion of cells with an ultrastructure consistent with healthy cells decreased from 70%, immediately after dissociation, to 30% after 8 h of incubation . Similar changes were also observed in cell suspensions treated with neuroprotectants . Thus, the neuroprotectants examined did not block the development of specific morphological signs of neurodegeneration . However, when also taking into account that dead cells lysed and disappeared from each cell suspension with time, we found that the total number of remaining viable cells with healthy nuclear chromatin or intact membrane integrity was significantly higher in the tirilazad mesylate-treated group . The results indicate that tirilazad mesylate protects only a small subpopulation of embryonic mesencephalic cells from degeneration induced by mechanical trauma during tissue dissection and dissociation.

Plant Cell Rep, 2003 Feb, 21(6), 503 - 10 Epub 2002 Dec 14.
Efficient callus formation and plant regeneration of goosegrass {Eleusine indica (L.) Gaertn.}; Yemets AI et al.; Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass { Eleusine indica (L.) Gaertn.} and its dinitroaniline-resistant biotypes . The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7 . The presence of organogenic and embryogenic structures in these calli was histologically documented . Cell suspension cultures derived from young calli were established in a liquid medium with the same composition . Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7 . Calli derived from the R-biotype of E . indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants . Embryogenic cell suspension culture was a better source of E . indica protoplasts than callus or mesophyll tissue . The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation . The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.

J Invest Dermatol, 2003 Jun, 120(6), 983 - 9
CX-659S, a diaminouracil derivative, indirectly inhibits the function of Langerhans cells by blocking the MEK1/2-Erk1/2 pathway in keratinocytes; Uchi H et al.; Keratinocytes are an important component of the skin immune system, and keratinocyte-derived cytokines control the function of Langerhans cells . We previously showed that CX-659S, a novel diaminouracil derivative, had an inhibitory effect on hapten-induced contact hypersensitivity reaction in mice . In this study, we investigated the mechanism by which CX-659S elicits its inhibitory effect . CX-659S inhibited the expressions of CD80 and CD86, but not that of CD54, on Langerhans cells in epidermal cell suspensions . Exogenous granulocyte-macrophage colony-stimulating factor restored the CX-659S-induced inhibition of CD80 and CD86 expressions of Langerhans cells . The production of interleukin-2 from allogeneic T cells was also inhibited when the cells were stimulated with CX-659S-treated epidermal cells, and this inhibition was suppressed by the addition of granulocyte-macrophage colony-stimulating factor during CX-659S treatment . As CX-659S significantly inhibited production of granulocyte-macrophage colony-stimulating factor from keratinocytes, CX-659S was thought to indirectly affect Langerhans cells by inhibiting the function of keratinocytes . These effects of CX-659S were preceded by blockade of the phosphorylation of extracellular-signal-regulated kinase 1/2 and their direct activators, mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2 (MEK1/2), but not p38 mitogen-activated protein kinase or inhibitory nuclear factor kappaBalpha, in keratinocytes . Furthermore, a specific MEK1/2 inhibitor, U0126, mimicked the effect of CX-659S . CX-659S, a keratinocyte-response modifier, would be an effective therapeutic compound to inhibit contact hypersensitivity reaction, its action mechanism being different from those of other immunosuppressive agents such as glucocorticosteroids or cyclosporine A.

Prep Biochem Biotechnol, 2003 May, 33(2), 87 - 100
Novel method for continuous cell separation by density gradient centrifugation: evaluation of a miniature separation column; Shiono H et al.; A compact bench-top model of the centrifuge enables continuous cell separation based on density differences . The apparatus holds a small separation disk equipped with a circular channel (8 mL capacity) separated by a septum . A set of isotonic Percoll media with different densities is continuously introduced at one terminal and collected from the other . Under a centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities . The performance of the apparatus was demonstrated with the separation of human buffy coat containing nucleated cells (>10(8)) among a large population (10(10)) of RBC . The results indicated that the method is capable of separating a large number of nucleated cells, with minimum damage, for a few hours of operation wherein neutrophils are well resolved from lymphocytes . The method may be applied to other types of samples including cord blood, blood from small animals, cultured cells, pancreatic beta cell islets, malaria parasites, sperm cells, etc.

Int J Dev Neurosci, 2003 Jun, 21(4), 191 - 8
Restorative potential of cholinergic rich transplants in cholchicine induced lesioned rats: a comparative study of single and multiple micro-transplantation approach; Agrawal AK et al.; Restorative potential of fetal neural transplantation in colchicine induced neurodegeneration was studied in rats; where colchicine (2.5mg per site) was administered bilaterally into the hippocampus followed by bilateral infusions of fetal neural cell suspension rich in cholinergic neurons as single macro- or multiple micro-transplants in the hippocampal region 3 weeks post-colchicine (2.5mg per site) lesion . Animals were studied for neuro behavioural and neurochemical recovery at 4 and 24 weeks post-transplantation and electrophysiological (single cell recording) and immunohistochemical parameters, choline acetyl transferase (ChAT) expression was studied in hippocampus at 24 weeks post-transplantation . Colchicine lesioned rats receiving single macro- or multiple micro-transplants exhibited significant restoration in cognitive dysfunction caused by colchicine after 4 weeks of transplantation which remain persistent in multiple micro-transplanted group upto 24 weeks post-transplantation, whereas, single macro-transplanted animals did not exhibit any significant recovery . Neurochemical studies revealed significant restoration in acetylcholine esterase activity and cholinergic (muscarinic) receptor binding after 24 weeks post-transplantation as compared to 4 weeks post-transplantation in multiple micro-transplanted group . Single cell recording studied at 24 weeks post-transplantation exhibited significant restoration in firing rates when compared with lesioned group . The viability of cholinergic fibre at transplanted sites has further been confirmed by increase in ChAT immuno positivity in hippocampal region using monoclonal antibody and quantified using image analyser Leica Qwin 500 software . The results suggest that intra-hippocampal multiple site cholinergic rich transplants provide better and long term restoration in the cholinergic deficits induced by colchicine lesion as compared to single site macro-transplantation.

J Urol, 2003 Jun, 169(6), 2316 - 9; discussion 2320
Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments: a pilot study; Hafez AT et al.; PURPOSE: We developed a cell transfer technology for covering demucosalized colonic segments with bladder urothelium . This covering would be achieved through aerosol spraying of single cell suspension of bladder urothelial and smooth muscle cells with fibrin glue onto the demucosalized colonic segments . MATERIALS AND METHODS: In 6 piglets (20 kg.) a 4 cm.2 area of bladder was excised . Single cell suspension of bladder urothelial and smooth muscle cells was prepared . A segment of detubularized sigmoid colon was isolated on its vascular pedicle and demucosalized . The single cell suspensions were combined with an equal volume of fibrin glue and sprayed over the raw submucosal surface of the sigmoid segment . The sigmoid segment was retubularized and sutured to the posterior peritoneum . Animals were sacrificed 4 weeks later, and the segment was submitted to histological and immunohistochemical analysis . RESULTS: Sigmoid segments appeared grossly intact with no reduction in surface area . Hematoxylin and eosin architecture revealed an intact urothelial layer . Deep to this layer was a randomly aligned but distinctly segregated layer of smooth muscle cells . The urological new smooth muscle layer stained positive for calponin and the urothelial layer was cytokeratin-7 and uroplakin III positive . CONCLUSIONS: Separation, cell suspension and aerosol delivery of bladder urothelial and smooth muscle cells in fibrin glue can successfully transfer these urological cell populations to a new host tissue commonly used in urological reconstruction . In vivo co-culture of bladder smooth muscle and urothelial cells results in coverage of a large area of demucosalized gut providing new potential for transfer and reconstitution of urologically functionally appropriate tissue to the bladder itself.

Protoplasma, 2003 May, 221(1-2), 93 - 100
Copper-mediated oxidative burst in Nicotiana tabacum L . cv . Bright Yellow 2 cell suspension cultures; Raeymaekers T et al.; In cell suspension cultures of Nicotiana tabacum L . cv . Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM) . This specific and early response towards copper stress was shown to be extracellular . Addition of 300 U of catalase per ml decreased the level of H(2)O(2) . Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2% . This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide . Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90% . The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.

Ann Hematol, 2003 Jul, 82(7), 435 - 9 Epub 2003 May 24.
Primary aleukemic myeloid leukemia cutis treated successfully with combination chemotherapy: report of a case and review of the literature; Chang H et al.; Aleukemic myeloid leukemia cutis is extremely rare and is usually associated with early marrow relapse and poor treatment outcome . We report a 39-year-old man presenting with generalized cutaneous nodules . The initial diagnosis was cutaneous malignant lymphoma . New skin lesions and a nasopharyngeal mass developed during phototherapy . Biopsy of the cutaneous and nasopharyngeal lesions revealed monotonous blast cell infiltration . Cytochemical stain and immunophenotypic analysis of the fresh cell suspension made from another skin biopsy specimen identified that the neoplastic cells belonged to the monocytic lineage . A diagnosis of primary aleukemic leukemia cutis was established . The leukemic cells expressed CD56 but did not carry AML-1/ETO, CBFbeta/MYH11, or common MLL fusion transcripts . He received standard induction therapy for acute myeloid leukemia, followed by high-dose postremission chemotherapy and has been disease-free for more than 30 months . To the best of our knowledge, the current case has the longest disease-free survival among those reported.

Int J Oral Maxillofac Surg, 2003 Jun, 32(3), 305 - 12
Influence of donor age and culture conditions on tissue engineering of mucosa autografts; Lauer G et al.; In oral surgery the transplantation of tissue engineered mucosa is used more frequently . The conventional single cell suspension culture method (SCSM) involves murine feeder cells and foetal calf serum . The explant technique (ET) has been used as alternative culture procedure . Aim was to study the efficacy of the ET and the SCSM without feeder cells to grow primary cultures and to test the effects of donor age, of extracellular matrix proteins (ECMP), and of autogenous serum on cell growth in explant cultures . These factors were assessed in cultures of 58 patients overall . In 48 cultures of 12 patients primary cell growth was compared between the ET and the SCSM . Eighteen of 24 cultures were established with the ET whereas only 3 of 24 were established with the SCSM . To test the influence of donor age on cell multiplication, the proliferation rate (DNA synthesis measured by bromodeoxyuridine uptake) and the overall growth (DNA content) was determined in cultures of five young and five old donors . In cultures from old donors (mean age 56 years) proliferation was lower but more sustained relative to the cultures from the young donors (mean age 25 years) . In old donors overall in vitro cell growth was only 2/3 of that in young donors . In cultures of 20 donors the influence on cell adhesion and growth of the ECMP fibronectin and laminin was assessed by planimetry . While ECMP augmented explant adhesion, these substances did not enhance keratinocyte growth significantly . Comparing the influence of autogenous and foetal calf serum on cell growth no differences were observed in all cultures of the six donors . In conclusion, the ET without additional ECMP coating and with autogenous instead of foetal calf serum are now used to culture gingival keratinocytes for tissue engineering mucosa grafts . Consequently xenogenous components are avoided, being a considerable advantage.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jun 25, 790(1-2), 317 - 25
Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography; Bermejo R et al.; B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae . Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics . Tedious methodologies for B-PE purification have been published . In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum . Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1) . After adsorption, washing was carried out in the expanded-bed mode . Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer . In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5 . With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies . The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.

J Gastrointest Surg, 2003 May-Jun, 7(4), 507 - 14; discussion 514-5
An intravital model to monitor steps of metastatic tumor cell adhesion within the hepatic microcirculation; Haier J et al.; Organ-specific tumor cell adhesion within the microcirculation of host organs is an important step in the metastatic cascade . Circulating tumor cells have to adhere within the microcirculatory vessels, quickly stabilize their adhesion and probably leave the circulation to avoid toxic effects of hydrodynamic shear forces of circulating blood . Using intravital fluorescence microscopy we established a new model for the intravital observation of colon carcinoma cell adhesion within the hepatic microcirculation . HT-29 (human) and CC531 (rat) colon carcinoma cells were fluorescence labeled using CalceinAM . Single cell suspensions were injected intraarterially in Sprague-Dawley rats . Using intravital fluorescence microscopy adhesive interactions of circulating tumor cells within the hepatic microcirculation were observed at the liver surface . These interactions were analyzed regarding their time course and the localization within the vascular tree . Autofluorescence of liver parenchyma was sufficient for distinction of hepatic sinusoids . Intravital microscopy enabled the differentiation of early events in adhesion formation within hepatic sinosoids, adhesion stabilization, and extravasation of the tumor cells into the liver parenchyma . Tumor cell adhesion occurred almost exclusively within sinusoidal capillaries; however, the diameter of these vessels was usually larger than that of the tumor cells leaving remaining perfused lumen of the capillaries . Colon carcinoma cells rapidly migrated into the liver parenchyma after successful adhesion within the sinusoids . In contrast to common endpoint assays of the metastatic cascade, this in vivo model allows investigations of metastatic colon carcinoma cell adhesion within the liver microcirculation as specific steps during the formation of hematogenous metastasis and their underlying mechanisms.

Infect Immun, 2003 Jun, 71(6), 3572 - 7
Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue; Shomer NH et al.; Susceptible strains of mice that are naturally or experimentally infected with murine intestinal helicobacter species develop hepatic inflammatory lesions that have previously been described as chronic active hepatitis . The inflammatory infiltrates in some models of chronic autoimmunity or inflammation resemble tertiary lymphoid organs hypothesized to arise by a process termed lymphoid organ neogenesis . To determine whether hepatic inflammation caused by infection with helicobacter could give rise to tertiary lymphoid organs, we used fluorescence-activated cell sorting, immunohistochemistry, and in situ hybridization techniques to identify specific components characteristic of lymphoid organs in liver tissue sections and liver cell suspensions from helicobacter-infected mice . Small venules (high endothelial venules {HEVs}) in inflammatory lesions in Helicobacter species-infected livers were positive for peripheral node addressin . Mucosal addressin cell adhesion molecule also stained HEVs and cells with a staining pattern consistent with scattered stromal cells . The chemokines SLC (CCL 21) and BLC (CXCL13) were present, as were B220-positive B cells and T cells . The latter included a naive (CD45lo-CD62Lhi) population . These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.

Free Radic Biol Med, 2003 Jun 1, 34(11), 1473 - 81
Spin traps: in vitro toxicity and stability of radical adducts; Khan N et al.; We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells . The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption) . We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells . Toxicity varied with the type of cell line and the parameter that was measured . In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3) . The radical adducts of the new spin traps have significantly increased stability as compared to DMPO . These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells . It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps . It then should be feasible to select the spin trap(s) best suited for the proposed study.

J Plant Physiol, 2003 Apr, 160(4), 339 - 46
Putrescine facilitated enhancement of capsaicin production in cell suspension cultures of Capsicum frutescens; Sudha G et al.; Putrescine treatment (0.1 mmol/L) influenced enhancement of growth and capsaicin production in the cell suspension cultures of C . frutescens . The administration of polyamine inhibitor DFMA (alpha-DL-difluoromethylarginine) resulted in a reduction of the growth, capsaicin content and the endogenous titres of polyamines (PAs) . The capsaicin synthase activity was also higher in the putrescine (Put) treated cultures . Ethylene levels were lower in the cultures treated with putrescine . This study suggested that Put facilitates growth and capsaicin production.

J Comp Physiol {B}, 2003 Jul, 173(5), 429 - 35 Epub 2003 May 20.
Activation of Na+/H+ exchange by protein phosphatase inhibitors in red blood cells of the frog Rana ridibunda; Gusev GP et al.; The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer . For this purpose the cells were treated with several known inhibitors of protein phosphatases . In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx . The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium . The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium . Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1) . However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors . The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content . Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride . The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride . Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A.

J Physiol, 2003 Jul 15, 550(Pt 2), 419 - 29 Epub 2003 May 16.
Aquaporin-1 and HCO3(-)-Cl- transporter-mediated transport of CO2 across the human erythrocyte membrane; Blank ME et al.; Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO3(-)-Cl- transporter may be involved in CO2 transport across biological membranes, but the physiological importance of this route of gas transport remained unknown . We studied CO2 transport in human red blood cell ghosts at physiological temperatures (37 degrees C) . Replacement of inert with CO2-containing gas above a stirred cell suspension caused an outside-to-inside directed CO2 gradient and generated a rapid biphasic intracellular acidification . The gradient of the acidifying gas was kept small to favour high affinity entry of CO2 passing the membrane . All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment . Inhibition of carbonic anhydrase (CA) demonstrated that CO2-induced acidification required the catalytic activity of CA . Blockade of the function of either AQP1 (by HgCl2 at 65 microM) or the HCO3(-)-Cl- transporter (by DIDS at 15 microM) completely prevented fast acidification . These data indicate that, at low chemical gradients for CO2, nearly the entire CO2 transport across the red cell membrane is mediated by AQP1 and the HCO3--Cl- transporter . Therefore, these proteins may function as high affinity sites for CO2 transport across the erythrocyte membrane.

Planta, 2003 Jul, 217(3), 498 - 506 Epub 2003 May 15.
Molecular characterization and post-transcriptional regulation of ME1, a type-I ribosome-inactivating protein from Mirabilis expansa; Vepachedu R et al.; Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thus arresting protein synthesis at the translocation step . In the present study, ME1, a type-1 RIP, was cloned and sequenced from storage roots of Mirabilis expansa (Ruiz & Pavon) . The full-length cDNA sequence of ME1 has 1,129 nucleotides with an open reading frame of 951 nucleotides representing 317 amino acids . Nucleotide analysis revealed that the N-terminal region of ME1 was cleaved, and the mature protein started at amino acid 34 . ME1 showed very close similarities to MAP and MAP-4 from Mirabilis jalapa . Southern blot analysis revealed the presence of two homologous genes for ME1 cDNA in M . expansa . Northern blot analysis showed high levels of ME1 transcripts in primary and storage roots . Interestingly, jasmonic acid induced ME1 transcript expression in cell suspension cultures of M . expansa; however, the production of ME1 protein was not enhanced as observed by Western blot analysis . Our data suggest that ME1 has the ability to depurinate its own mRNA, thus inhibiting its translation . These observations suggest a possible mechanism by which ME1 protein levels are post-transcriptionally regulated.

J Plant Physiol, 2003 Mar, 160(3), 227 - 37
Coumarin inhibits the growth of carrot (Daucus carota L . cv . Saint Valery) cells in suspension culture; Abenavoli MR et al.; We used a carrot (Daucus carota L . cv . Saint Valery) cell suspension culture as a simplified model system to study the effects of the allelochemical compound coumarin (1,2 benzopyrone) on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates . Exposure to micromolar levels of coumarin caused severe inhibition of cell growth starting from the second day of culture onwards . At the same time, the presence of 50 mumol/L coumarin caused accumulation of free amino acids and of ammonium in the cultured cells, and stimulated their glutamine synthetase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxylase activities . Malate dehydrogenase, on the other hand, was inhibited under the same conditions . These effects were interpreted in terms of the stimulation of protein catabolism and/or interference with protein biosynthesis induced by coumarin . This could have led to a series of compensatory changes in the activities of enzymes linking nitrogen and carbon metabolism . Because coumarin seemed to abolish the exponential phase and to accelerate the onset of the stationary phase of cell growth, we hypothesise that such allelochemical compounds may act in nature as an inhibitor of the cell cycle and/or as a senescence-promoting substance.

Biochem J, 2003 Aug 15, 374(Pt 1), 51 - 61
P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx; Amstrup J et al.; P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas . Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown . In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells . We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+ . EGFP-P2X7 receptors localized to the plasma membrane, clusters within the membrane and intracellularly . Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP . Using C- and N-terminal P2X7-receptor mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation . On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants . In cell suspensions prepared from rat pancreas we show that P2X7 receptors also activate ERK1 and ERK2, indicating that these signalling pathways are also turned on in native epithelium.

Mol Plant Microbe Interact, 2003 May, 16(5), 456 - 64
Nonspecific lipid-transfer protein genes expression in grape (Vitis sp.) cells in response to fungal elicitor treatments; Gomes E et al.; Nonspecific lipid transfer proteins (nsLTPs) are small, basic cystein-rich proteins believed to be involved in plant defense mechanisms . Three cDNAs coding nsLTPs from grape (Vitis vinifera sp.) were cloned by reverse-transcriptase-polymerase chain reaction (RT-PCR) and PCR . The expression of nsLTP genes was investigated in 41B-rootstock grape cell suspension, in response to various defense-related signal molecules . Ergosterol (a fungi-specific sterol) and a proteinaceous elicitor purified from Botrytis cinerea strongly and rapidly induced the accumulation of nsLTP mRNAs . Jasmonic acid, cholesterol, and sitosterol also promoted nsLTPs mRNA accumulation, although to a lesser extent, whereas salicylic acid had no effect . High performance liquid chromatography analysis indicated that the amounts of three LTP isoforms (previously named P1, P2, and P4) were increased by ergosterol . None of the four isoforms displayed any significant antifungal properties, with the exception of the P4 isoform, which reduced Botrytis mycelium growth in vitro, but only in calcium-free medium . The results are discussed in the context of plant-pathogen interactions.

Tissue Eng, 2003 Apr, 9(2), 315 - 25
Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh; van den Dolder J et al.; The objective of the present study was to learn more about the effect of seeding and loading techniques on the osteogenic differentiation in vitro of rat bone marrow cells into titanium fiber mesh . This material was used as received or subjected to glow discharge treatment (RFGD) . The seeding methods that were used included a so-called droplet, cell suspension (high and low cell density), and rotating plate method . Osteogenic cells were cultured for 4, 8, and 16 days into titanium fiber mesh . DNA, osteocalcin, scanning electron microscopy (SEM) analysis, and calcium measurements were used to determine cellular proliferation and differentiation . DNA analysis of the differently seeded specimens showed that proliferation proceeded faster in the first versus second run for droplet and cell suspension samples . No clear and distinct additional effect was found when RFGD treatment was used . Statistical analyses revealed that high cell density and low rotational speed resulted always in a significantly higher DNA content . Calcium measurements and osteocalcin analysis showed that using high cell densities during inoculation of the scaffolds prevented the occurrence of differences between experimental runs . SEM examination showed that for droplet and cell suspension samples cells were present at only one side of the mesh . The mesh side where the cell sheet was observed depended on the additional use of glow discharge treatment . On these materials, the cells had penetrated through the meshes and formed a cell sheet at the bottom side . When rotation was used, no cell sheet was formed and cells had invaded the meshes and were growing around the titanium fibers . On the basis of our results, we conclude that (1) . titanium fiber mesh is indeed suitable to support the osteogenic expression of bone marrow cells, and (2) . changing the initial cell density as well as the use of dynamic seeding methods can influence the osteogenic capacity of the scaffold.

Biol Pharm Bull, 2003 May, 26(5), 642 - 50
Oxidosqualene cyclases from cell suspension cultures of Betula platyphylla var . japonica: molecular evolution of oxidosqualene cyclases in higher plants; Zhang H et al.; Betula platyphylla var . japonica is a rich source of triterpenoid as it contains dammarane type triterpenes in the leaves, and lupane type and oleanane type triterpenes in the bark . Four oxidosqualene cyclase cDNAs (BPX, BPX2, BPW and BPY) were cloned by homology based PCR methods from cell suspension cultures of B . platyphylla var . japonica . Open reading frames consisting of 2274, 2304, 2268 and 2340 bp were ligated into yeast expression plasmid pYES2 under the control of GAL1 promoter and introduced into lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77 . Analyses of in vitro enzyme activities and/or accumulated products in the transformants demonstrated that they encode cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and beta-amyrin synthase (BPY) proteins . Phylogenetic tree was constructed for all the known oxidosqualene cyclases (OSCs) including the clones obtained in this study, revealing that OSCs having the same enzyme function form respective branches in the tree even though they derive from different plant species . Intriguing correlation was found between reaction mechanism and molecular evolution of OSCs in higher plants.

Cancer Res, 2003 May 1, 63(9), 1999 - 2004
Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles; Sokolov K et al.; Recent developments in photonic technology provide the ability to noninvasively image cells in vivo; these new cellular imaging technologies have the potential to dramatically improve the prevention, detection, and therapy of epithelial cancers . Endoscope-compatible microscopies, such as optical coherence tomography and reflectance confocal microscopy, image reflected light, providing a three-dimensional picture of tissue microanatomy with excellent spatial resolution (1-10 micro m) . However, their ability to image molecular biomarkers associated with cancer is limited . Here, we describe a new class of molecular specific contrast agents for vital reflectance imaging based on gold nanoparticles attached to probe molecules with high affinity for specific cellular biomarkers . The application of gold bioconjugates for vital imaging of precancers is demonstrated using cancer cell suspensions, three-dimensional cell cultures, and normal and neoplastic fresh cervical biopsies . We show that gold conjugates can be delivered topically for imaging throughout the whole epithelium . These contrast agents have potential to extend the ability of vital reflectance microscopies for in vivo molecular imaging . They can potentially enable combined screening, detection, and therapy of disease using inexpensive imaging systems; such tools could allow mass screening of diseases such as cancer in resource-poor settings.

Transfus Apheresis Sci, 2003 Jun, 28(3), 319 - 27
Filtration induces changes in activity states and leucocyte populations; Trindade H et al.; The distribution of leucocyte subpopulations in platelet concentrates (PC) derived from pre-storage filtered platelet-rich plasma (PRP), the cell suspension obtained by reverse filter washing and the post-filtered PC, were monitored by immunophenotyping analysis using CD3, CD20 and CD33 . Leucocyte activation analysis with the CD11b marker revealed that this molecule is up regulated in neutrophils taken from the filter . This, together with the loss of cell viability during the enrichment process, suggests that contact with the filter matrix and processing and storage of samples containing leucocytes may lead to activation and loss of leucocyte viability . These changes were found to be more pronounced in less stable myeloid cells and account for the differences reported among various authors which in some cases related to operational conditions such as the enrichment process used and the length of time between filtration and analysis of samples . Finally, statistical analysis of the results obtained by immunophenotypic studies indicate that post-filter samples (S) contain significantly higher numbers of CD33+ myeloid cells when compared to (PF) the pre-filter samples (65.03%+/-12.6 and 24.56%+/-14.73, p<0.0000), with a decrease in T cells (50.72%+/-14.80 in PF and 24.05+/-9.48 in the cell suspension (S), p<0.0007) and B cells (14.96+/-9.31 in PF and 9.9+/-5.22 in S, p<0.201) . A new strategy for assessing the influence of the filtration process on residual leucocyte activation and viability is described . This has direct relevance to collection, processing, storage and quality monitoring of PC.

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