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,