Phytochemistry, 2000 Sep, 55(1), 51 - 7 Antifungal monoterpene production in elicited cell suspension cultures of Piqueria trinervia; Saad I et al.; Cell suspension cultures of the traditional medicinal plant Piqueria trinervia Cav., which synthesizes monoterpene piquerol A, were established . A defense response was induced in the cultures when eight homogenized fungi isolated from wild populations of P . trinervia were added . Piquerol A was not produced in the elicited system, while four other substances were synthesized de novo . They were excreted into the medium and inhibited in vitro fungal growth . The most abundant substance produced in this system was a new monoterpene: 2-methylene-7,7-dimethylbicyclo (3,3,1) heptane-4,6-diol . Monoterpenes in the cell suspension culture reported here were produced via two metabolic channels: the first acted constitutively and expressed in liquid and solid cultures, the second is inducible in response to several pathogens and elicitor substances. FEBS Lett, 2000 Sep 29, 482(1-2), 125 - 30 Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells; Seregelyes C et al.; We have isolated a 483-bp-long full-length cDNA clone encoding a non-symbiotic hemoglobin called Mhb1, the first one found in alfalfa . This non-symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome . The Mhb1 mRNA was found only in the roots of alfalfa plants . The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment . The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture . The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm . A potential link to the nitric oxide signalling pathway is also discussed. Biochim Biophys Acta, 2000 Sep 27, 1487(2-3), 113 - 27 Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo; Kamat JP et al.; Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties . Its antioxidant ability, however, has not been examined in detail . Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions . Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls . Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances . The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase . ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride . Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM . The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol . When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis . To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen . The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1) . In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS. Hum Pathol, 2000 Sep, 31(9), 1051 - 4 Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping; Bavikatty NR et al.; CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML) . However, until recently, analysis for CD10 has generally required fresh or frozen tissue . 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue . Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL . CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias . The results matched in 100% (20 of 20) of AML . Five percent (1 of 20) of AMLs expressed CD10 . Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry . CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL . Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry . In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry . We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia . This technique is useful in distinguishing AML from ALL. Arthritis Rheum, 2000 Sep, 43(9), 2046 - 55 Mesenchymal cells expressing bone morphogenetic protein receptors are present in the rheumatoid arthritis joint; Marinova-Mutafchieva L et al.; OBJECTIVE: To evaluate the presence of cells of an early mesenchymal lineage, as judged by the expression of bone morphogenetic protein receptors (BMPRs), in the joints of normal individuals and patients with rheumatoid arthritis (RA) . METHODS: Synovial fluids, single cell suspensions of cultured fibroblast-like synoviocytes (FLS), and synovial tissues were examined by immunohistology with antibodies to BMPR type IA (BMPRIA), BMPRIB, and BMPRII and then quantified using computerized image analysis . Other antibodies were evaluated by cytofluorography . RESULTS: In primary cultures of joint effusions from patients with RA and other forms of inflammatory arthritis, there were large adherent cells with the appearance of either fibroblasts or stromal cells that stained with antibodies to mesenchymal elements-CD44, type I collagen, alpha-actin, and vimentin-but not with antibodies to hematopoietic markers . These cells proliferated rapidly, expressed BMPRIA and BMPRII, and soon became the predominant cells in culture . They were retained through multiple passages and persistently displayed surface vascular cell adhesion molecule 1 . Immunohistochemical analysis of cultured RA FLS (passages 3, 4, and 6; n = 6) revealed that 11.6% were BMPR-positive, while only 2.0% of osteoarthritis FLS (passage 4; n = 3) were BMPR-positive, and 1 normal synovial culture had no BMPR-positive cells . In all RA synovial membranes examined (n = 9), BMPRI- and BMPRII-expressing cells were identified in the intimal lining and were also scattered in the subintima . These cells constituted approximately 25% and approximately 7% of the cells in each area, respectively . Double immunostaining showed no coexpression of BMPR-positive cells with CD68, CD34, or CD3 . Cells expressing BMPR were not seen in any normal synovial samples (n = 4) . Strong staining for BMPR was identified on cells at the invasive front of the pannus and at sites of cartilage erosion . CONCLUSION: The inflamed RA joint contains BMPR-positive mesenchymal cells . Their origin is still speculative, but since their counterparts in the bone marrow are essential for osteoclastogenesis, support lymphocyte development and maturation, and protect T cells and B cells from programmed cell death, the BMPR-positive cells may be essential elements in the pathogenesis of RA and other inflammatory forms of chronic synovitis. Exp Cell Res, 2000 Oct 10, 260(1), 146 - 59 Stem cell characteristics of transplanted rat mammary clonogens; Kim ND et al.; Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials . When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient . Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens . Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition . The current experiments were designed to test these expectations . The data are discussed in the context of results of previous studies with this and other experimental models . The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells . Dig Dis Sci, 2000 Aug, 45(8), 1631 - 8 Reaggregation of rat dissociated myenteric plexus in extracellular matrix gels; Schafer KH et al.; The aim of this study was to investigate the growth behavior of freshly dissociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurotrophic factor (GDNF) . Therefore, cell suspensions of the dissected myenteric plexus of newborn rats were cultured in freshly prepared gels of commercially available mixtures of collagen, laminin, and hepatoglycans as a first step towards mimicking the natural environment of the myenteric plexus . The cultures were kept either in chemically defined serum-free medium alone or supplemented with GDNF . Cultures on polylysinc-coated glass cover slips served as controls . Dissociated myenteric plexus grown on polylysine formed dense clusters of neurons with radially outgrowing nerve fibers, while the neurons cultured in the gel reaggregated to much smaller clusters . These contained, depending on the culture conditions, 2-10 neurons . The morphology of the network that was seen in the gels after a few days in vitro resembled very closely the in situ situation of the submucous plexus and the myenteric plexus in hypoganglionic children . Electron microscope investigations showed a high degree of organization with fiber bundles and vesicle-containing varicosities and growth cones . Independent of the method of culturing, GDNF obviously influenced the growth behavior of the dissociated plexus . The size of the ganglia was larger, and the secondary network denser when GDNF was supplemented . Moreover, the enteric neurons in the gel cultures tended to be larger in size when treated with GDNF . Three-dimensional cultures of dissociated myenteric plexus in an ECM gel might be a valuable tool towards the understanding of the formation of the enteric nervous system during development, especially considering pathological conditions such as Hirschsprung's disease or other dysganglionic diseases. Pflugers Arch, 2000, 440(5 Suppl), R193 - 4 Effect of pH on red blood cell deformability; Kuzman D et al.; The effect of pH on the red blood cell (RBC) deformability, which is a consequence of a change of cell membrane elastic properties is studied experimentally . With the intention to reduce the effects on deformability of cell geometry and cytoplasmic viscosity, we measured the deformability of the cells with the same volume at various pH of cell suspension from 6.2 to 8.0 . Constant cell volume was achieved by varying osmolarity . Deformability was quantified by measuring the elongation of RBCs subjected to velocity gradient in a transparent cone-plate rheoscope . Observed significant decrease of deformability at lower pH leads to the conclusion that membrane elastic properties could be affected by pH changes in the range from 6.2 to 8.0. Pflugers Arch, 2000, 440(5 Suppl), R49 - 50 In vitro functional tests for evaluation of stimulating capacity of cultured human dendritic cells; Hajdinjak T et al.; Basic functional test for evaluation of in vitro cultured human dendritic cells (DC) is primary allogeneic one-way mixed lymphocyte reaction (MLR) . In this way, one can evaluate stimulating capacity, which is a basic characteristic of DC . The proliferation of cells is measured through incorporation of 3H-thymidine . Normally proliferation is measured at days 5-7 . We studied kinetics of proliferative responses initiated with different stimulating cell suspensions to evaluate differences and possibly reduce time needed to perform this test . Gradual increase in response from days 1 to 7 and a significant difference from controls (peripheral blood mononuclear cells) seen from day 4 was noted if macrophages were used as stimulators . A consistently higher proliferation, compared to controls, was always found already on day 2 when mature DC were used as stimulators . The reaction peaked 2 to 3 days earlier and was also more than two times more intense . This maximal and significantly higher response, consistently seen already after 48 hours, allows us to confirm the presence of mature DC in stimulating suspensions much earlier than previously. Plant J, 2000 Sep, 23(6), 785 - 94 Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity; Orvar BL et al.; Many plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non-freezing temperatures at the onset of winter . CA is associated with gene expression that requires transient calcium influx into the cytosol . Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold-acclimatize . Conversely, chemical agents causing increased calcium influx induce cold acclimatization-specific (cas) gene expression in alfalfa at 25 degrees C . How low temperature triggers calcium influx is, however, unknown . We report here that induction of a CA-specific gene (cas30), calcium influx and freezing tolerance at 4 degrees C are all prevented by cell membrane fluidization, but, conversely, are induced at 25 degrees C by membrane rigidification . cas30 expression and calcium influx at 4 degrees C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25 degrees C by the actin microfilament destabilizer cytochalasin D (CD) . JK blocked the membrane rigidifier-induced, but not the calcium channel agonist-induced cas30 expression at 25 degrees C . These findings indicate that cytoskeleton re-organization is an integral component in low-temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidification and calcium influx in CA. J Invest Dermatol, 2000 Oct, 115(4), 680 - 6 Magnesium ions inhibit the antigen-presenting function of human epidermal Langerhans cells in vivo and in vitro . Involvement of ATPase, HLA-DR, B7 molecules, and cytokines; Schempp CM et al.; The combination of seawater baths and solar radiation at the Dead Sea is known as an effective treatment for patients with psoriasis and atopic dermatitis . Dead Sea water is particularly rich in magnesium ions . In this study we wished to determine the effects of magnesium ions on the capacity of human epidermal Langerhans cells to stimulate the proliferation of alloreactive T cells . Twelve subjects were exposed on four subsequent days on the volar aspects of their forearms to 5% MgCl2, 5% NaCl, ultraviolet B (1 minimal erythemal dose), MgCl2 + ultraviolet B, and NaCl + ultraviolet B . Epidermal sheets were prepared from punch biopsies and were stained for ATPase and HLA-DR . Compared with untreated skin, the number of ATPase+/HLA-DR+ Langerhans cells was significantly reduced after treatment with MgCl2 (p = 0.0063) or ultraviolet B (p = 0.0005), but not after NaCl (p = 0.7744) . We next questioned whether this reduced expression of ATPase and HLA-DR on Langerhans cells bears a functional relevance . Six subjects were treated on four subsequent days with 5% MgCl2, ultraviolet B (1 minimal erythemal dose), and MgCl2 + ultraviolet B . Epidermal cell suspensions from treated and untreated skin were assessed for their antigen-presenting capacity in a mixed epidermal lymphocyte reaction with allogeneic naive resting T cells as responder cells . Treatment with MgCl2, similarly to ultraviolet B, significantly reduced the capacity of epidermal cells to activate allogeneic T cells (p = 0.0356) . Magnesium ions also suppressed Langerhans cells function when added to epidermal cell suspensions in vitro . The reduced antigen-presenting capacity of Langerhans cells after treatment with MgCl2 was associated with a reduced expression by Langerhans cells of HLA-DR and costimulatory B7 molecules, and with a suppression of the constitutive tumor necrosis factor-alpha production by epidermal cells in vitro . These findings demonstrate that magnesium ions specifically inhibit the antigen-presenting capacity of Langerhans cells and may thus contribute to the efficacy of Dead Sea water in the treatment of inflammatory skin diseases. Eur J Ultrasound, 2000 Sep, 12(1), 81 - 8 Mechanical properties of bovine aortic endothelial cells in suspension studied by ultrasonic interferometry; Boynard M et al.; OBJECTIVE: Cell adhesion phenomenon has been extensively studied in the last decade and was shown to be mediated by specialized molecules and driven by physical forces . Cohesion of the vessel wall cells is also dependent on adhesion molecules but less is known about the physical forces involved . To investigate endothelial cell/endothelial cell interaction from a mechanical point of view, we have used an ultrasonic interferometry device, named EchoCell, which has been previously designed to study red blood cell-red bood cell (RBC-RBC) interaction . METHODS: Bovine aortic endothelial (BAE) cells were cultured, detached, then suspended in buffer and their mechanical and geometrical properties studied with the EchoCell system . The ultrasonic apparatus measures both the accumulation rate of cells in suspension on a solid plate and the acoustical impedances of the suspension and the sediment . RESULTS: In suspension, BAE exhibited, in our experimental conditions (3x10(6) cells per ml), a spherical size evaluated by calculation at a mean radius of 7+/-2 microm . Moreover, no BAE aggregation occurred at the concentrations used . The acoustical impedance of the BAE suspensions calculated from all the samples studied, in the cell concentration range from 1.5x10(6) to 6x10(6) cells per ml, was 1.52x10(6) Rayl (kg m(-2) s(-1)) . Furthermore, the acoustical impedance of the cell sediment was found to be independent on the initial cell suspension concentration and equal to 1.63x10(6) Rayl (kg m(-2) s(-1)) . Estimation of the volume fraction of BAE inside the sediment allows to evaluate the ultrasonic velocity and the elastic bulk modulus of cells . CONCLUSION: The ultrasonic interferometry method appears particularly interesting to study geometrical and mechanical (acoustical impedance, sound velocity, elastic bulk modulus) properties of BAE cells. Ultrasound Med Biol, 2000 Jul, 26(6), 1043 - 9 High-frequency backscatter and attenuation measurements of selected bovine tissues between 10 and 30 MHz; Maruvada S et al.; There are now diagnostic ultrasonic imaging devices that operate at very high frequencies (VHF) of 20 MHz and beyond for clinical applications in ophthalmology, dermatology, vascular surgery, endoluminal imaging and small animal imaging . To be able to better interpret these images and to further the development of these devices, knowledge of ultrasonic attenuation and scattering of biological tissues in this frequency range is crucial . Attenuation and backscatter coefficients (BSCs) of bovine tissues in the frequency range of 10 to 30 MHz were measured, respectively, using a standard substitution method for attenuation measurements and a modified narrow-band substitution method for scattering measurements . A modified substitution method for scattering measurements has to be used at high frequencies because unfocused transducers due to their decreased sensitivity cannot be used in the simple substitution method . In the modified method, the flat reflector is substituted by a particulate reference medium whose BSC is well-known and documented; in this case, a red cell suspension . In this paper, experimental results on BSC and attenuation coefficient measured between 10 and 30 MHz are reported . The frequency dependence of backscatter of the selected bovine tissues ranges from 2.4 to 3.5, whereas attenuation is observed to be still approximately linearly proportional to frequency . The BSC measured with the modified method is in good agreement with those obtained with the standard method between 10 and 20 MHz. Plant Sci, 2000 Sep 8, 158(1-2), 129 - 137 Decrease in the regeneration potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration by the auxin transport inhibitor, 2,3,5-triiodobenzoic acid; Nakano M et al.; Cell suspension cultures of Lilium formosanum Wallace were initiated from bulb scale-derived calli and subcultured every 2 weeks using a medium containing 5 microM 4-amino-3,5,6-trichloropicolinic acid (picloram) . Almost all cell clumps from the suspension cultures developed numerous somatic embryos following their transfer onto a plant growth regulator-free medium, while they vigorously produced shoot buds on media containing 0.5 or 5 microM 6-benzyladenine (BA) . The high regeneration potential on a plant growth regulator-free medium was maintained for up to 54 months, but it gradually decreased thereafter, and only a few adventitious shoots and embryos were obtained from 75-month-old cultures . For restoring the regeneration potential of these cultures, various treatments with plant growth regulators were applied, among which about 10-fold increases in the number of regenerated shoot buds were obtained with 0.5 or 5 microM 2,3,5-triiodobenzoic acid (TIBA) in combination with 0.5 or 5 microM BA or N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea (thidiazuron) . Only shoot buds were produced from the cell clumps cultured on TIBA-containing media, and these shoot buds developed into complete plantlets after they were excised from the calli and transferred to a plant growth regulator-free medium. Plant Sci, 2000 Sep 8, 158(1-2), 41 - 51 Oligosaccharides potentiate methyl jasmonate-induced production of paclitaxel in Taxus canadensis; Linden JC et al.; The interdependence of methyl jasmonate (MJ) with chitin and chitosan derived elicitors in formation of paclitaxel was studied using plant cell suspension cultures of Taxus canadensis . Induction of paclitaxel biosynthesis was enhanced when MJ and elicitors were added 8 days after culture transfer compared to treatments in which only MJ or only elicitors were added . The enhancement of the paclitaxel biosynthesis response to MJ concentration was roughly linear between 0 and 200 microM using colloidal chitin or oligosaccharides of chitin and chitosan as elicitors . MJ concentrations greater than 200 microM were inhibitory . In kinetic studies, culture growth and substrate utilization were inhibited when the cultures were elicited with 100 microM MJ and with 0.63 mg l(-1) N-acetylchitohexaose and with 100 microM MJ alone; paclitaxel yields were 10-fold greater under the latter condition than the former . Ethylene biosynthesis by the cell cultures in response to elicitation is implicated in regulation of the response. Prikl Biokhim Mikrobiol, 2000 Jul-Aug, 36(4), 422 - 7 {Proteolytic activity of the enzyme complex of the mammalian pancreas in comparison with pancreatin}; Berdutina AV et al.; The proteolytic activity and thermal stability of the enzyme complex of cell suspension from pig and bovine pancreas glands was compared with those of pancreatin . The enzyme complex displayed the highest thermal stability and activity at 50 degrees C . The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 +/- 0.1) at which this complex had the maximum proteolytic activity were determined . Pancreatin had a pH optimum of 8.0 +/- 0.1. Exp Neurol, 2000 Oct, 165(2), 268 - 77 Time course of apoptotic cell death within mesencephalic cell suspension grafts: implications for improving grafted dopamine neuron survival; Sortwell CE et al.; The vast majority ( congruent with 90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum . Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation . This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts . Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively . Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum . Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation . However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days . No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found . Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis . Photochem Photobiol, 2000 Sep, 72(3), 320 - 6 A novel express bioassay for detecting toxic substances in water by recording rhodopsin-mediated photoelectric responses in Chlamydomonas cell suspensions; Govorunova EG et al.; The influence of Cu2+, Zn2+, Cd2+, Pb2+ and formaldehyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was investigated using three modifications of a recently developed population method for electrical recording (in nonoriented, phototactically preoriented (PO) and gravitactically preoriented cell suspensions) . The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the responses . Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simultaneously with an inhibition of their photoelectric cascade, photo-orientation and motility . The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive phototaxis in the presence of the toxic substances . Of the two major components of the photoelectric cascade, the regenerative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the photoreceptor current . The results obtained show that measurement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bioassay for testing environmental pollutants in water samples. Planta, 2000 Aug, 211(3), 430 - 9 Molecular characterization of the expression of distinct classes of cyclins during the early development of tomato fruit; Joubes J et al.; Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds from two distinct phases of growth, essentially cell division and cell expansion . In this study, we investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins, during tomato fruit development . We isolated six genes designated Lyces;CycA1;1, Lyces;CycA2;1, Lyces; CycA3;1, Lyces;CycB1:1 and Lyces;CycB2;1 encoding tomato mitotic cyclins, and Lyces;CycD3;1 encoding a G1 cyclin . The accumulation of transcripts was predominantly associated with mitotically active organs: developing fruits, young leaves and roots, and with cell-suspension cultures under appropriate sugar feeding conditions . Transcripts for all the isolated cyclin genes could be detected in the epidermis and pericarp of fruit tissues where some slight mitotic activity still remained at the onset of ripening . However, Lyces;CycA3;1 and Lyces;CycD3;1 were expressed in the gel tissue at the late stage of fruit development, suggesting that they are involved in endoreduplication of the differentiated and giant cells of the gel tissue. J Immunol Methods, 2000 Aug 28, 242(1-2), 21 - 31 Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization; Curry MP et al.; Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs . To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays . The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue . The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects . Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques . A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I . These cells were suitable for phenotypic characterisation by flow cytometry . They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines. Med Biol Eng Comput, 2000 Jul, 38(4), 384 - 9 Derivation of extracellular fluid volume fraction and equivalent dielectric constant of the cell membrane from dielectric properties of the human body . Part 2: A preliminary study for tracking the progression of surgical tissue injury; Tatara T et al.; A study is conducted to determine whether the extracellular fluid (ECF) volume fraction and equivalent dielectric constant of the cell membrane epsilon m, derived from the dielectric properties of the human body can track the progression of surgical tissue injury . Frequency-dependent dielectric constants and electrical conductivities of body segments are obtained at surgical (trunk) and non-surgical sites (arm and leg) from five patients who have undergone oesophageal resections, before and at the end of surgery and on the day after the operation . The ECF volume fraction and the equivalent epsilon m of body segments are estimated by fitting the dielectric data for body segments to the cell suspension model incorporating fat tissue, and their time-course changes are compared between body segments . By the day after the operation, the estimated ECF volume fraction has increased in all body segments compared with that before surgery, by 0.13 in the arm, 0.16 in the trunk and 0.14 in the leg (p < 0.05), indicating postoperative fluid accumulation in the extracellular space . In contrast, the estimated equivalent epsilon m shows a different time course between body segments on the day after the operation, characterised by a higher change ratio of epsilon m of the trunk (1.34 +/- 0.66, p < 0.05), from that of the arm (0.66 +/- 0.34) and leg (0.61 +/- 0.11) . The results suggest that the equivalent epsilon m of a body segment at a surgical site can track pathophysiological cell changes following surgical tissue injury. Med Biol Eng Comput, 2000 Jul, 38(4), 377 - 83 Derivation of extracellular fluid volume fraction and equivalent dielectric constant of the cell membrane from dielectric properties of the human body . Part 1: Incorporation of fat tissue into cell suspension model in the arm; Tatara T et al.; The non-invasive characterisation of cell pathophysiology is clinically important . A cell suspension model is applied to derive the extracellular fluid (ECF) volume fraction and the equivalent dielectric constant of the cell membrane epsilon m from the dielectric properties of human arms . Frequency-dependent dielectric constants and electrical conductivities of arms are obtained from 35 surgical patients over a frequency range of 5-1000 kHz . The cell suspension model is applied to fit the data using a complex non-linear least-squares method . The arms show typical dielectric dispersions, although the cell suspension model yields a poor fitting in dielectric constants at lower frequencies and electrical conductivities at higher frequencies . In contrast, a new cell suspension model, taking into account the fat tissue component, remarkably improves the overall fitting performance, allowing estimation of the volume fractions of ECF (0.34 +/- 0.05) and fat tissue (0.16 +/- 0.04) and the equivalent epsilon m (23 +/- 9) . The resulting estimates of the volume fraction of fat tissue are in good correlation with arm skinfold thickness (fat volume fraction of arm = 2.42 x 10(-3) x arm skinfold thickness (mm) + 0.099, R = 0.756, p < 0.0001) . Therefore it is concluded that the newly derived cell suspension model is well suited for the description of the dielectric properties of human tissues and thus the derivation of the ECF volume fraction and equivalent epsilon m. Biocell, 2000 Aug, 24(2), 139 - 44 Cell lines of Taxus species as source of the anticancer drug taxol; Goleniowski ME; Callus culture of Taxus baccata and Taxus x Media were induced using explants of young stems and female gametophyte . Culture conditions have been established for the cell suspension of the different callus cell lines . Callus were induced from Taxus baccata and Taxus x Media using Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4 D (2,4-dichlorophenoxyacetic) and benzylaminopurine (BA) . All the cultures grew slowly following the first subculture and the majority turned brown and ceased growth . The fast growing callus lines constituted a habituated callus lines (CFGTB; CFGTM; CSTB and CSTB) . These callus lines were used to induce cell suspension in the best nutritional medium (1 mg/l 2,4-D and 0.1 mg/l BA) . The callus exhibited levels of taxol ranging from 0.1 to 15 mg/Kg on a dry weight basis . Suspension cultures of Taxus baccata (CSTB and CFGTB) and Taxus x Media (CSTM and CFGTM) were maintained at 25 degrees C on a MS medium with two weeks transfers . The maximum taxol production for suspension cell was within the range 5 to 6 mg/l. Biocell, 2000 Aug, 24(2), 133 - 8 Peroxidases from cell suspension cultures of Brassica napus; Agostini E et al.; Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators . They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots . Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures . Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme . Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points . These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research. Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 179 - 82 {Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion}; Guo WW et al.; Protoplasts isolated from cell suspension cultures of 'Bonnaza' navel orange (Citrus sinensis L . Osbeck) were electrically fused with mesophyll protoplasts of rough lemon (Citrus jambhiri Lush) and Goutou orange (Citrus aurantium L.) respectively . Plants regenerated from both fusion combinations . Chromosome counting of randomly selected fifty two globular embryoids as well as all the regenerated seventy four plants from Bonnaza navel + rough lemon revealed that twenty six embryoids were tetraploids, and the rest were diploids while 100% regenerated plants were tetraploids . The results inferred that somatic hybrids were more competitive than parental genotypes in the process of plant regeneration . All the regenerated 14 plants from Bonnaza navel + Goutou orange were tetraploids as revealed by chromosome counting . POX isozyme and RAPD analysis verified that the plants from Bonnaza navel + rough lemon were hybrids, and RAPD analysis confirmed the hybridity of those from Bonnaza navel + Goutou orange. Phytochemistry, 2000 Aug, 54(7), 657 - 66 Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis; Warzecha H et al.; Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds . In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides . Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l . Cell cultures do contain a specific glucosidase . known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis . Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli . RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized . The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed . Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells. Phytochemistry, 2000 Aug, 54(7), 649 - 55 Flavanone 8-dimethylallyltransferase in Sophora flavescens cell suspension cultures; Yamamoto H et al.; Dimethylallyl diphosphate: naringenin 8-dimethylallyltransferase (EC 2.5.1) was characterized . The enzyme was enantiospecific for (-)-(2S)-naringenin and utilized 3,3-dimethylallyl diphosphate as sole prenyl donor . It required Mg2+ (optimum concentration, 10 mM), and has an optimum pH of 9-10 . The apparent Km values for 3,3-dimethylallyl diphosphate and naringenin were 120 and 36 microM, respectively . The microsomal fraction prenylated several other flavanones at the C-8 position less effectively as compared with naringenin . Interestingly, when 2'-hydroxynaringenin was used as a prenyl acceptor, the 8-lavandulyl (sophoraflavanone G) and the 6-dimethylallyl derivatives were formed, together with the 8-dimethylallyl derivative, leachianone G . These results suggest that the 2'-hydroxy group of naringenin plays an important role for the formation of a lavandulyl group. Braz J Med Biol Res, 2000 Sep, 33(9), 1015 - 21 A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system; Yarzabal A et al.; The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied . Plasminogen bound to the surface of H . pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA . Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins . Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension . The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells . epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors . The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established . Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated . These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages. Cell Transplant, 2000 May-Jun, 9(3), 395 - 407 The morphology, integration, and functional efficacy of striatal grafts differ between cell suspensions and tissue pieces; Watts C et al.; In order to develop a surgical protocol for use in clinical trials of striatal transplantation in Huntington's disease (HD), the issues involved in the preparation and implantation of the embryonic striatal tissue must be addressed . Rodent models of HD offer the best experimental paradigm with which to study various aspects of striatal transplantation . In this article we present the results of an investigation of the role of trypsin and the process of trituration in the preparation of cell suspensions compared to the use of solid pieces of tissue . The embryonic material was derived from the lateral ganglionic eminence (LGE) and implanted into the excitotoxically lesioned striatum of the host rats . Twelve weeks following implantation, retrograde tracing of projections from the graft to the globus pallidus was performed . Grafts derived from cell suspensions triturated in the presence of trypsin contained larger quantities of striatal tissue within the graft and more DARPP-32-positive medium spiny neurons than grafts implanted as fragments of tissue . Afferent and efferent connectivity was also better in the trypsinized suspension graft group . Modest recovery in paw reaching was observed contralateral to the grafted side in animals implanted with solid fragments of embryonic striatal tissue . No relationship was observed between functional effect and the graft anatomy . These results suggest that local graft host interaction may also be involved in graft-mediated functional recovery. Dermatology, 2000, 201(1), 15 - 20 Changes in keratin 6 and keratin 10 (co-)expression in lesional and symptomless skin of spreading psoriasis; Mommers JM et al.; BACKGROUND: Keratin 6 (K6) and keratin 10 (K10) are markers for epidermal hyperproliferation and differentiation, respectively, and are both expressed in the suprabasal layers of the epidermis . They may be co-expressed in different stages of the spreading psoriatic lesion, but single expression can also occur . OBJECTIVE: To investigate to what extent keratinocytes express K6 and K10, and to what extent they co-express K6 and K10 in different stages of the psoriatic lesion . We studied this in spreading psoriatic plaques . METHODS: Three 3-mm punch biopsies were obtained from the inner involved margin of a spreading lesion, from the uninvolved skin immediately adjacent to the spreading plaque, and from the distant uninvolved skin of 8 patients with incipient psoriasis . From 9 healthy volunteers, 3-mm punch biopsies were obtained as controls . After preparation of single cell suspensions of these biopsies, a triple staining protocol was performed with markers for K6 (monoclonal antibody LHK6B), K10 (monoclonal antibody RKSE60) and DNA content (TO-PRO-3 iodide) . Subsequently, cells were measured with a flow cytometer and the proportion of the markers was calculated using specific software . RESULTS: We observed a population of K6/K10-co-expressing cells, but also populations expressing only K6 . These subpopulations varied with the involvement of the lesion . There was a statistically significant difference between the inner margin and the outer margin with respect to the proportion of K6- and K10-expressing cells, whereas more K6-positive and K10-negative cells were detected in the inner margin of the lesions . The proportion of K6/K10-co-expressing cells in the inner margin was significantly different from the distant uninvolved skin . CONCLUSION: We confirmed that individual keratinocytes in psoriasis can express K6 or K10 depending on their localization in involved or uninvolved skin . There is a unique subpopulation of cells in the psoriatic plaques which co-express K6 and K10 . More studies are required to fully understand the pathogenic relevance of co-expression and single expression of K6 and K10 . Folia Histochem Cytobiol, 2000, 38(3), 129 - 31 Encapsulation of parathyroid cells in hollow fibers: a preliminary report; Granicka LH et al.; The purpose of experiments was to evaluate the survival and functioning of human parathyroid cells after encapsulation in hollow fibers (HFs) . The polypropylene HFs K600(PP Accurel (Akzo-Nobel, Germany) of inner diameter 0.6 mm, wall thickness 0.2 mm, original or surface modified were used for encapsulation . Production of parathormone (PTH) by encapsulated cells was measured in vitro . HF were filled with parathyroid cell suspension and tightly closed . Encapsulated cells were cultured for 9 or 33 days in RPMI 1640 containing 10% FCS or in Chang's medium . The level of PTH, produced by encapsulated cells was evaluated in the culture medium with radioimmunoassay test (RIA) . The assays were performed every 2-4 days . The result of PTH assay was similar in both types of tested media as well as with unmodified and modified HFs, being 2-4 pg/ml of culture medium per 10(3) encapsulated cells . In conclusion, encapsulation in original or modified HFs ensures diffusion of nutrients from culture medium to encapsulated cells and allows for functioning of cells for at least 33 days in vitro. Br J Ophthalmol, 2000 Sep, 84(9), 1004 - 7 Topical ophthalmic beta blockers may cause release of histamine through cytotoxic effects on inflammatory cells; van Beek LM et al.; AIM: To evaluate the effects of beta blockers used in ophthalmology on the release of histamine from mixed cell preparations containing human leucocytes and basophils . METHODS: A mixed leucocyte and basophil preparation was obtained from venous blood of healthy non-atopic volunteers . Cell preparations were then incubated with betaxolol, metipranolol, timolol, or carteolol . After incubation for 1 hour the histamine content of the supernatant was analysed by automated fluorometric analysis . Cell viability was tested by measuring lactate dehydrogenase (LDH) concentrations . RESULTS: Betaxolol and metipranolol in concentrations between 10(-2) M and 10(-3) M liberated histamine from human blood cells in a dose dependent manner . Carteolol and timolol had no effect on histamine at these concentrations . At the same concentrations LDH was also detected in the supernatants of cell suspensions incubated with metipranolol or betaxolol . CONCLUSIONS: Betaxolol and metipranolol induce substantial histamine release from human leucocytes, probably as a result of their cytotoxic effect. Jpn J Cancer Res, 2000 Aug, 91(8), 853 - 8 The combined effect of electroporation and borocaptate in boron neutron capture therapy for murine solid tumors; Ono K et al.; 10 B-Enriched borocaptate (BSH) was administered intraperitoneally to SCCVII tumor-bearing C3H / He mice . Electroporation (EP) was conducted by using a tweezers-type electrode . The (10) B contents in tumors were measured by prompt gamma-ray spectrometry . The colony formation assay was applied to investigate the antitumor effects of boron neutron capture therapy (BNCT) and thereby to estimate the intratumor localization of BSH . The (10) B concentrations in tumors decreased with time following BSH administration, falling to 5.4(0 . 1) ppm at 3 h, whereas EP treatment (3 repetitions) 15 min after BSH injection delayed the clearance of BSH from tumors, and the (10) B level remained at 19.4(0.9) ppm at 3 h . The effect of BNCT increased with the (10) B concentration in tumors, and the combination with EP showed a remarkably large cell killing effect even at 3 h after BSH injection . The effect of BNCT, i.e., slope coefficient of the cell survival curve of tumors, without EP was proportional to tumor (10) B level (r = 0.982), and that of BSH-BNCT combined with EP lay close to the same correlation line . However, tumors subjected to EP after BSH injection did not show high radiosensitivity when irradiated after conversion to a single cell suspension by enzymatic digestion . This indicates that the increase of the BNCT effect by EP was a consequence of enclosure of BSH in the interstitial space of tumor tissue and not within tumor cells . This is different from a previous in vitro study . The combination of EP and BNCT may be clinically useful, if a procedure to limit EP to the tumor region becomes available or if an alternative similar method is employed. Xenobiotica, 2000 Jul, 30(7), 665 - 81 Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes; Hewitt NJ et al.; 1 . The effect of cryopreservation on the metabolic capacity of monkey hepatocytes over 4 h in suspension and 24 h in culture was determined . Hepatocytes were diluted in a buffer containing 10% DMSO and frozen in a computer-controlled chamber . 2 . Initial ethoxyresorufin and ethoxycoumarin O-deethylase (ECOD) activities were the same in fresh and cryopreserved (CP) hepatocytes . ECOD activity in suspensions declined over 4 h but was the same in fresh and CP hepatocytes . 3 . The formation of testosterone hydroxy (OHT) metabolites (namely 6beta-OHT, 2beta-OHT, 16beta-OHT, 16alpha-OHT, 15beta-OHT, 2alpha-OHT and 6beta-OHT) was unaffected by cryopreservation . The loss of OHT activities over 4 h in CP and fresh whole cell suspensions was attributed to a loss of cofactor . CP hepatocyte cultures had equivalent OHT activities to freshly isolated hepatocytes . 4 . Initial UDP-glucuronyltransferase (UGT) activities, using the substrates 4-methylumbelliferone, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP whole hepatocytes . At later times, UGT activity was lower in CP than fresh hepatocytes but this was due to a loss of UDPGA . Initial sulphotransferase (SULT) activities, using the substrates 2-naphthol, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP hepatocytes . SULT activities were less stable than UGT activities but were the same in fresh and CP hepatocytes throughout the 4-h incubation . 5 . Initial glutathione S-transferase activities (using 1-chloro-2,4-dinitrobenzene) were the same in fresh and CP hepatocytes and both did not decrease over 4 h . 6 . CP monkey hepatocytes are a useful model for metabolic and cytotoxicity studies . These cells can be can be used either in suspension or in culture. Radiat Res, 2000 Sep, 154(3), 301 - 6 Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis; Holl V et al.; The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood . As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro . BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy . Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days . Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide . It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons . Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2 . However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture . The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations . Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons. Cas Lek Cesk, 2000 May 24, 139(10), 305 - 8 {Determination of intracellular molecules in cells in hematopoietic malignancy using flow cytometry}; Touskova M et al.; BACKGROUND: Detection of membrane molecules by immunophenotypisation is a routine counterpart of the laboratory tests which are needed for the complex diagnostic procedures of hematopoetic malignancies at present . The immunochemical detection of intracellular molecules is essential in such cases, where the expression of surface molecules on malignant cells is poor, or where the results of other diagnostic analyses are unequivocal (cases with ectopic expression of other lineage-specific molecules and cases of biphenotypic leukemias) . METHODS AND RESULTS: We followed the procedure using 4% solution of paraformaldehyd in phosphate buffered saline for fixation of cells . Lysing solution G (Becton-Dickinson) was used for permeabilisation of cells to detect nuclear and cytoplasmatic molecules . The permeabilised cell suspension was than washed with 0.5% solution of Tween 20 in phosphate buffered saline . This simple and rapid procedure is readily available for the routine detection of intracellular molecules . This approach is important for the diagnosis of hematopoetic malignancies. Anticancer Res, 2000 Jul-Aug, 20(4), 2773 - 8 Flow cytometric analysis of antigen expression in malignant and normal renal cells; Li G et al.; Flow cytometry allows quantitative analysis of cancer cells . The aim of this study was to make a quantitative study of antigen expression in malignant and normal renal cells in order to find the efficient monoclonal antibodies (mAbs) for labelling renal cancer cells . MATERIAL AND METHODS: 15 malignant and adjacent normal renal tissues and three renal carcinoma cell lines (ACHN, A704 and CAKI-2) were analyzed . The malignant and normal renal tissues were dissociated mechanically into cell suspension . The mAbs and isotype controls were used for immunochemical labelling . The stained cells were analyzed by flow cytometry . RESULTS: Renal tumor associated antigen G 250 was frequently detected in malignant renal cells but not in normal renal cells . Renal tumor associated antigen gp200 recognized by 66.4.C2 and PN-15 was frequently detected in malignant cells, normal renal cells and also in all three carcinoma cell lines . Epithelial antigens were strongly positive in normal renal cells . Compared with MOC 31, Ber-EP4 and E 29, W-lD9 was mostly reactive to malignant renal cells . VU-1D9 was strongly positive on ACHN and A704 . The carbohydrate carcinoma antigens CA 125, DF3 and Sialyl Lewis(a) were detectable in some of the malignant and normal renal cells . Sialyl Lewis(a) could be weakly detected on ACHN and A 704 . Pan-cytokeratins and cytokeratin (CK) 8 were strongly expressed in malignant and normal renal cells and in all three cell lines . CONCLUSION: Our results indicated that G 250, 66.4.Ca, PN-15, VU-1D9, MNF116 and anti-ckg were efficient mAbs for labelling renal cancer cells . Their potential clinical application by flow cytometry should be explored. J Biomed Mater Res, 2000 Nov, 52(2), 346 - 53 Microfabricated elastomeric stencils for micropatterning cell cultures; Folch A et al.; Here we present an inexpensive method to fabricate microscopic cellular cultures, which does not require any surface modification of the substrate prior to cell seeding . The method utilizes a reusable elastomeric stencil (i.e., a membrane containing thru holes) which seals spontaneously against the surface . The stencil is applied to the cell-culture substrate before seeding . During seeding, the stencil prevents the substrate from being exposed to the cell suspension except on the hole areas . After cells are allowed to attach and the stencil is peeled off, cellular islands with a shape similar to the holes remain on the cell-culture substrate . This solvent-free method can be combined with a wide range of substrates (including biocompatible polymers, homogeneous or nonplanar surfaces, microelectronic chips, and gels), biomolecules, and virtually any adherent cell type . J Biomed Mater Res, 2000 Nov, 52(2), 246 - 55 Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy; Ponticiello MS et al.; Adult mesenchymal stem cells (MSCs), found in the bone marrow, have the potential to differentiate into multiple connective tissue types, including cartilage . In this study, we examined the potential of a porous gelatin sponge, Gelfoam, for use as a delivery vehicle for MSCs in cartilage regeneration therapy . Adult human MSCs (hMSCs) were seeded throughout the gelatin sponge after a 2-h incubation period . When cultured for 21 days in vitro in a defined medium supplemented with 10 ng/mL of TGF-beta 3, hMSC/Gelfoam constructs produced a cartilage-like extracellular matrix containing sulfated glycosaminoglycans (s-GAGs) and type-II collagen, as evident upon histologic evaluation . Constructs loaded with a cell suspension of 12 x 10(6) cells/mL produced an extracellular matrix containing 21 microg of s-GAG/microg of DNA after 21 days of culture . This production was more efficient than constructs loaded at higher or lower cell densities, indicating that the initial seeding density influences the ability of cells to produce extracellular matrix . When implanted in an osteochondral defect in the rabbit femoral condyle, Gelfoam cylinders were observed to be very biocompatible, with no evidence of immune response or lymphocytic infiltration at the site . Based on these observations we conclude that Gelfoam resorbable gelatin sponge is a promising candidate as a carrier matrix for MSC-based cartilage regeneration therapies . Br J Dermatol, 2000 Aug, 143(2), 307 - 12 A comparative study of Fas and Fas-ligand expression during melanoma progression; Soubrane C et al.; BACKGROUND: Impaired regulation of apoptosis is known to be associated with the development of various cancers . Fas receptor (APO-1/CD95) binding to its ligand, Fas-ligand (Fas-L), has been shown to trigger apoptosis in various cell types . OBJECTIVES: In this study, we examined CD95 and Fas-L expression on primary and metastatic melanoma cells from patients to investigate a potential correlation between these measures of apoptosis and different disease stages . PATIENTS AND METHODS: Primary melanoma cells were obtained after surgical resection from 19 patients and metastatic cells from fine-needle aspiration of lymph nodes or palpable subcutaneous lesions in 25 patients . Normal skin cells were obtained at skin biopsy of 10 healthy donors . RESULTS: Flow cytometric analysis revealed that CD95 and Fas-L expression was detected in all the kinds of cell studied . In whole cell suspensions, CD95 expression was significantly higher (P < 0.0001) in normal skin cells than in melanoma cells, whatever the stage studied . By contrast, we observed an increase in Fas-L expression in melanoma cells compared with normal ones . Subsequently, using a double staining method, we studied these measures on HMB45+ cells, a specific marker for melanoma cells, and found that CD95 expression was significantly higher (P = 0.0005) in primary than in metastatic cells while Fas-L expression was significantly increased (P = 0 . 0004) in metastatic compared with primary cells . Furthermore, a relationship was found between CD95 or Fas-L expression and Breslow thickness; as primary melanoma thickness progressively increased, the percentage of HMB45+ CD95+ cells decreased while that of HMB45+ Fas-L+ cells concurrently increased . CONCLUSIONS: These results suggest that downregulation of CD95 and upregulation of Fas-L in melanoma might be considered as concomitant with disease progression. Lab Invest, 2000 Aug, 80(8), 1215 - 25 Culture of dendritic cells from a nonlymphoid organ, the thyroid gland: evidence for TNFalpha-dependent phenotypic changes of thyroid-derived dendritic cells; Croizet K et al.; Because they are sparsely distributed in tissues, dendritic cells (DC) present in nonlymphoid organs are difficult to isolate . Only DC from skin and lung have been successfully studied in culture . The objective of the present work was to investigate the possibility of isolating and culturing DC from an endocrine organ, the thyroid gland, which is particularly susceptible to the development of autoimmune processes . The study was conducted on pig thyroid glands to have sufficient amounts of starting material . This choice required the characterization of immunological reagents capable of recognizing DC markers in the pig species . Using a discontinuous trypsinization procedure, a DC population representing 2% to 3% of the thyroid cell suspension was reproducibly obtained . Isolated DC quantitatively attached to tissue culture-treated dishes and segregated from thyrocytes . DC identified as cells expressing major histocompatibility complex class II molecules, the mannose receptor, and the S100 protein were found to have a high capacity to internalize labeled ligands, dextran, and mannosylated albumin . These cells had a phenotype of immature DC . Secondarily, a fraction of DC detached from culture dishes, and floating DC had low or no endocytic activity, a characteristic of mature DC . Treatment of DC/thyrocytes cocultures with tumor necrosis factor alpha (TNFalpha) activated the transformation of immature DC into mature DC . These data show that DC isolated from the thyroid gland can be maintained immature or activated to undergo maturation in primary culture . The procedure of cell isolation and culture should be adaptable to human thyroid tissue for in vitro analyses of DC-mediated immune responses. Exp Dermatol, 2000 Aug, 9(4), 266 - 70 Flow-cytometric characterization of normal versus psoriatic epidermis using improved cell separation methodology; Seegers BA et al.; Recently a new approach for epidermal cell characterization was developed: three-parameter flow cytometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin . The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin . Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions . The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin . The new methodology revealed data with a narrow range . In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation. Exp Dermatol, 2000 Aug, 9(4), 240 - 7 Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro; Laihia JK et al.; It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation . Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function . In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo . To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion . The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR . The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later . The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen . HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo . Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro . We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon. Shock, 2000 Aug, 14(2), 144 - 9 Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma; Hasko G et al.; Polyamines are endogenous immunomodulatory molecules . Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide . In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions . Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages . The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected . In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production . The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice . The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase . In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody . In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10. Int J Radiat Biol, 2000 Aug, 76(8), 1129 - 41 Exposure of human osteosarcoma and bone marrow cells to tumour-targeted alpha-particles and gamma-irradiation: analysis of cell survival and microdosimetry; Aurlien E et al.; PURPOSE: This study was designed to compare the cytotoxic effects of an alpha-emitting radioimmunoconjugate, which binds to osteosarcoma but not to bone marrow cells, with those of external gamma-irradiation . MATERIALS AND METHODS: The human osteosarcoma cell line, OHS-s1, and mononuclear cells from bone marrow (BM) harvested from healthy donors, were used for these experiments . Cells in suspension were added to various activity concentrations of the anti-osteosarcoma monoclonal antibody TP-3 radiolabelled with 211At . Following incubation for 1 h, unbound radioactivity was washed off and cell survival was determined from clonogenic assays . Microdosimetry was calculated based on binding and retention kinetics of 211At to the cells, as well as cellular and nuclear diameters . For comparison, cell suspensions were irradiated with a single dose of 60Co gamma-rays . RESULTS: 211At-labelled TP-3 showed heterogeneous binding to OHS-s1 cells, with a considerable variation among experiments . About 78% of the initially bound 211At decayed while associated with the OHS-s1 cells . D0 values estimated by microdosimetry were 0.33 (0.22-0.48, range) Gy and 1.18 (0.89-1.89) Gy for OHS-s1 and BM cells, respectively, whereas D0 values after external beam irradiation were 0.86+/-0.07Gy and 1.71+/-0.22Gy . The relative biological effectiveness (RBE) of 211At-labelled TP-3 at 37% survival was 3.43 for OHS-s1 and 1.55 for BM . CONCLUSIONS: High-LET targeted alpha-particle exposure killed osteosarcoma cells more effectively than bone marrow cells, although heterogeneous antigen expression among these tumour cells limited the magnitude of this effect. Am J Clin Pathol, 2000 Aug, 114(2), 258 - 63 Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20; Cornfield DB et al.; The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic . We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments . Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining . CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression . In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH . This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH . The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question . Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined. J Exp Bot, 2000 Apr, 51(345), 807 - 15 Limiting CO2 levels induce a blue light-dependent HCO3- uptake system in Monoraphidium braunii; Giraldez N et al.; The in situ photoactivation of an HCO3- uptake system in the green alga Monoraphidium braunii requires the irradiation of the cell suspensions with short wavelength radiation (blue, UVA and/or UVC) . Plasma membrane ATPase inhibitors block the uptake of this monovalent anion at pH 9 . M . braunii cells grown in high CO2 lack an HCO3- uptake system in their plasma membrane, but those grown in low CO2 can take up this anion at high rates . Cells grown in high CO2, transferred to CO2-limiting conditions in the light, start taking up HCO3- in 30 min, although they take 90 min to reach maximum rates of HCO3- transport . Therefore, this induction process seems to be triggered by low external CO2 concentration . In fact, increasing or decreasing the external HCO3- concentration does not induce the uptake system and only a decrease in CO2 concentration in the medium triggers the induction process . The appearance of the HCO3- transport activity is sensitive to cycloheximide, indicating that cytoplasmic protein biosynthesis is necessary for the induction of the uptake system . Photosynthetically active radiation, but not particularly blue light, is essential for induction of the uptake system to occur and the inhibition of photosynthesis by DCMU blocks it . From these results it can be inferred that when M . braunii cells detect a drop in CO2 concentration, they induce a blue light-dependent HCO3- uptake system. J Exp Bot, 2000 Apr, 51(345), 685 - 93 Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture; Kawano T et al.; Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L . cv . Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration . Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS . The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction . The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected . Similar results were obtained using HRP . It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro . The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals . The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2 . In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions . The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide. J Biomed Opt, 2000 Apr, 5(2), 131 - 7 Light scattering from cells: the contribution of the nucleus and the effects of proliferative status; Mourant JR et al.; As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei . The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells . Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures . An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined . Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells . Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus . This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells . Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria. Fish Shellfish Immunol, 2000 Jan, 10(1), 21 - 31 Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures; Diago ML et al.; This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium . When TPS cultures were kept in the same flask, i.e . without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells . Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells . Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers . The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane . Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions . Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium . These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions. Plant Sci, 2000 Jul 28, 156(2), 185 - 192 Comparison of the phytotoxic activity of the phytotoxin destruxin B and four natural analogs; Pedras MS et al.; A quantitative bioassay utilizing staining of plant cell suspension cultures of Sinapis alba was employed to establish a structure-phytotoxic activity correlation among destruxin B, homodestruxin B, and desmethyldestruxin B, toxins produced by Alternaria brassicae (Berk.) Sacc., the causative agent of Alternaria blackspot of brassicas . In addition, the phytotoxicity of destruxin B, homodestruxin B, and their respective metabolites hydroxydestruxin B and hydroxyhomodestruxin B were tested on resistant and susceptible plant species utilizing in planta leaf assays and leaf uptake of toxin solutions . Overall, the results obtained from punctured leaf and cell staining assays indicated that homodestruxin B (EC(50) 3x10(-4) M) was the most toxic of the five compounds, followed by destruxin B (EC(50) 5x10(-4) M), and desmethyldestruxin B (EC(50)&z.Gt;5x10(-4) M) . On the other hand, the hydroxylated destruxins (hydroxydestruxin B EC(50)&z.Gt;5x10(-4) M) were significantly less phytotoxic than the parent toxins. Biotechnol Prog, 2000 Jul-Aug, 16(4), 668 - 70 Nutrient medium optimization for rosmarinic acid production by Lavandula vera MM cell suspension; Pavlov AI et al.; The overall effect of NH(4)NO(3), KNO(3), and KH(2)PO(4) on the biosynthesis of rosmarinic acid and cell biomass by Lavandula vera MM cell suspension was studied by the method of the full factor experiment . Polynomial regression models were elaborated to give a quantitative description of the processes of biosynthesis of rosmarinic acid (Y(1)) and cell biomass (Y(2)) as a result of the variation of the concentration of NH(4)(+), 0.09 g/L < or = X(1) < or = 0 . 23 g/L; NO(3)(-), 2.44 g/L < or = X(2) < or = 3.02 g/L; and KH(2)PO(4), 0 . 170 < or = X(3) < or = 0.425 g/L . Optimization procedures according to the modified Simplex method allowed us to establish the optimal conditions for the biosynthesis of rosmarinic acid by Lavandula vera MM: X(1*) = 0.09 g/L; X(2*) = 3.02 g/L, and X(3*) = 0.170 g/L, where Y(1)max = 1786.74 mg/L (27 times higher compared with the cultivation in the standard Linsmayer-Skoog medium) . As a result, modified ingredients of the Linsmayer-Skoog nutrient medium were applied for the cultivation of Lavandula vera MM to achieve a maximum yield of rosmarinic acid. Transplantation, 2000 Jul 27, 70(2), 327 - 35 Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations; Kung L et al.; BACKGROUND: The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency . Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency . In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA . METHODS: We compared their effects on NFATC2 dephosphorylation using Western analysis, interferon-gamma production using ELISA, and CN phosphatase activity using the CN assay in human peripheral blood leukocytes (PBL) and mouse spleen cell suspension . RESULTS: The FK concentration inhibiting 50% (IC50) of all three activities was approximately 0.2 microg/ml in human PBL, versus 5-20 microg/ml for CsA . Although inhibition of interferon-gamma secretion and NFATC2 dephosphorylation was complete, inhibition of CN phosphatase activity was incomplete with both drugs at saturation, particularly with FK . Inhibition of CN phosphatase activity was incomplete whether FK treatment was in vivo in mouse or in vitro in various human and mouse tissues, especially brain . Exogenous FKBP12 or CyPA increased CN phosphatase inhibition, suggesting that incomplete inhibition of CN phosphatase activity reflected limiting amounts of active immunophilin . CONCLUSIONS: These data contradict the prevailing assumption that immunophilins are abundant and not limiting for inhibition of CN by CsA or FK . Further, the observation that FK and CsA completely inhibit immune function without completely inhibiting CN suggests that the inhibition of immune function is not mediated by general CN inhibition but by inhibition of a subset of CN which is critical for lymphocyte activation. Eur J Biochem, 2000 Aug, 267(16), 5005 - 13 Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture; Sasabe M et al.; We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans . INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment . INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively . To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses . The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases . These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells. Clin Exp Allergy, 2000 Aug, 30(8), 1166 - 71 Diagnosis of venom allergy by flow cytometry . Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release; Sainte-Laudy J et al.; BACKGROUND: Potent allergens such as hymenoptera venoms are capable of inducing severe and life threatening clinical reactions . Percentage of false negative results obtained by the usual diagnostical methods is comprised between 10 and 25% . OBJECTIVE: Evaluation of the sensitivity and the specificity of cellular tests and particularly evaluation of a new flow cytometric method . METHODS: Forty-five allergic patients having experienced a local, a systemic reaction or an anaphylactic shock and 10 controls having undergone hymenoptera stings without clinical reactions were selected on the basis of the clinical history, skin tests and specific IgE . Three cellular tests were performed on the same cell suspensions and in the presence of 2 ng/mL of rIL3: histamine release (RIA), leukotriene C4 release (ELISA) and basophil activation test (flow cytometry after double anti-IgE FITC, anti-CD63 PE labelling) . RESULTS: As compared to the clinical history, sensitivities of skin tests, specific IgE, flow cytometry, histamine release and leukotriene release were, respectively; 85%, 88%, 100%, 89% and 100% . Flow cytometric analysis of basophil activation showed a significant decrease of the mean fluorescence density and number of IgE positive cells and a significant increase of the number of CD63 positive cells . The 10 controls tested by flow cytometry were negative . CONCLUSION: As compared to the clinical history and to the other parameters tested here, flow cytometry showed a high sensitivity and a high specificity . The excellent correlation observed between this method and the other cellular tests such as histamine and leukotriene release are in favour of the specificity of flow cytomery and in favour of the use of this method for venom allergy diagnosis. Magn Reson Imaging, 2000 Jul, 18(6), 689 - 95 Effects of cell volume fraction changes on apparent diffusion in human cells; Anderson AW et al.; Diffusion-weighted imaging was used to study the relationship between apparent diffusion coefficient (ADC) and cell volume fraction in cell suspensions and packed arrays . Cell volume fraction was varied by changing extracellular fluid osmolarity (for human glial cells) and by changing cell density (for human glial and red blood cells) . In packed arrays of glial cells, ADC increased 10% when cells shrank and decreased 13% when cells swelled . ADC decreased 34% as cell density increased from 0 to 72% . In erythrocyte suspensions, ADC decreased 90% as the cell density increased from 0 to 89% . These results agree with theoretical predictions. Biochem J, 2000 Aug 15, 350 Pt 1, 215 - 8 Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals; Gieseg S et al.; This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species . U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups . In an incubation for 22 h, 1.2 mM peroxyl radicals was generated and each cell acquired 1.5x10(8) -OOH groups . These groups were located on the cell proteins; no lipid peroxidation was detected . The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals . There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins . The half-life of protein hydroperoxides in cell suspensions was approx . 4 h at 37 degrees C . Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species. Cryobiology, 2000 Jun, 40(4), 360 - 9 Green fluorescent protein: A novel viability assay for cryobiological applications; Elliott G et al.; Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ . Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly . Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues . As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability . To the authors' knowledge, this is the first use of GFP in cryobiology applications . Intracellular GFP fluorescence was evaluated following slow freezing . Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath . Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr) . A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97 . A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay . Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions . The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate . Gene Ther, 2000 Aug, 7(15), 1259 - 64 Assessing gene expression in vivo: magnetic resonance imaging and spectroscopy; Bell JD et al.; Recent developments in magnetic resonance imaging and spectroscopy afford the possibility of detecting and assessing transfer, expression and subsequent therapeutic changes of effector or marker transgenes noninvasively . In the field of MR imaging, 'smart' MR contrast agents are being developed, so called because they change their conformational structure and in so doing induce MR detectable changes in a given tissue . These agents become 'switched on' in response to physiological changes brought about by the enzymatic action of a given gene product (enzymes), and are being developed for use in intact cells, isolated organs and animal models . Ultimately, these agents hold the promise of bridging the gap between the laboratory and the patient with noninvasive detection of transgene expression in vivo in man . Similarly, magnetic resonance spectroscopy is being developed as a noninvasive method to assess transgene expression indirectly by means of MR visible intracellular markers . These markers take the form of intracellular endo/exogenous metabolites associated with exogenous enzyme expression and function . Again, this technique will be applicable to a variety of different situations, from cell suspensions through to clinical imaging of the whole body . In this article the unique opportunities for laboratory-based and clinical studies afforded by MR techniques are discussed. Cytometry, 2000 Aug 1, 40(4), 327 - 35 Ultraviolet-induced detection of halogenated pyrimidines: simultaneous analysis of DNA replication and cellular markers; Hammers HJ et al.; BACKGROUND: We describe a new nonenzymatic methodology that allows the simultaneous detection of DNA replication and other cellular markers such as immunophenotyping . DNA replicating cells are identified by their incorporation of halogenated thymidine analogs, e.g., 5-bromo-deoxyuridine (BrdUrd) . METHODS: Irradiation with ultraviolet (UV)-B or UV-A light in the presence of Hoechst 33258 and subsequent treatment with a hypotonic buffer makes BrdUrd accessible to monoclonal antibodies (mAb), thus allowing its sensitive detection . RESULTS: The photolysis of BrdUrd in DNA with UV light is sequence dependent and results in DNA damage, allowing the detection of remaining BrdUrd using hypotonic conditions . However, treatment with other inducers of single or double- strand breaks of DNA such as gamma irradiation or hydrogen peroxide did not allow BrdUrd detection . The new methodology is compatible with both mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i.e., alcohols . The successful identification of CD34+, CD138+, or CD19+ cells out of heterogeneous cell suspensions and their cell-cycle analysis are described . Results correlated very well with acid denaturation (r = 0.972) . The average coefficient of variation (CV) of G(1) in the DNA histogram was smaller than 5%, resulting in good preservation of DNA distribution . Also, the signal-to-noise ratio was almost twice as high as for 2N acid denaturation, facilitating convenient discrimination of BrdUrd-positive cells . CONCLUSIONS: In contrast to previous approaches, this methodology eliminates the need for any additional enzymatic treatment such as DNA digestion or strand-break labeling after UV irradiation . The method is fast, convenient, and inexpensive and should be able to promote the use of halogenated pyrimidines in basic and clinical research of cancer, immunology, and pharmacology . Cancer, 2000 Jul 15, 89(2), 288 - 96 Thymidylate synthase quantitation and in vitro chemosensitivity testing predicts responses and survival of patients with isolated nonresectable liver tumors receiving hepatic arterial infusion chemotherapy; Link KH et al.; BACKGROUND: Patients with isolated, nonresectable liver tumors may receive regional hepatic arterial infusion (HAI) chemotherapy with response rates of about 50% . The objective of this study was to investigate the value of thymidylate synthase (TS) determination in combination with in vitro chemosensitivity testing to predict the responses and survival of patients receiving HAI . METHODS: TS mRNA expression was quantitated using a reverse transcription-polymerase chain reaction technique with beta-actin as the internal standard . In vitro chemosensitivity testing was performed with tumor cell suspensions using the human tumor colony-forming assay (HTCA) . RESULTS: An analysis of the test combination in 24 consecutive patients revealed that 77% (10 of 13) of the sensitive and 9% (1 of 11) of the resistant patients had complete or partial clinical responses . Sensitive patients were 8.5-fold more likely to respond (P = 0.0036) and displayed with 32 months (range, 5-75 months) a longer median survival than resistant patients with 17 months (range, 3-28 months, P = 0.003) . Analysis of the Kaplan-Meier curves revealed that sensitive patients had a higher overall survival probability, as determined by the log rank test (P = 0.044) . CONCLUSIONS: These results suggest that the clinical outcomes of patients receiving HAI therapy may be predictable with TS quantitation and HTCA . It is possible, therefore, that this combination may be used in the future to select patients with liver tumors who will benefit from HAI before the start of regional chemotherapy . Am J Physiol Heart Circ Physiol, 2000 Aug, 279(2), H657 - 71 Estimating oxygen transport resistance of the microvascular wall; Vadapalli A et al.; The problem of diffusion of O(2) across the endothelial surface in precapillary vessels and its utilization in the vascular wall remains unresolved . To establish a relationship between precapillary release of O(2) and vascular wall consumption, we estimated the intravascular flux of O(2) on the basis of published in vivo measurements . To interpret the data, we utilized a diffusion model of the vascular wall and computed possible physiological ranges for O(2) consumption . We found that many flux values were not consistent with the diffusion model . We estimated the mitochondrial-based maximum O(2) consumption of the vascular wall (M(mt)) and a possible contribution to O(2) consumption of nitric oxide production by endothelial cells (M(NO)) . Many values of O(2) consumption predicted from the diffusion model exceeded M(mt) + M(NO) . In contrast, reported values of O(2) consumption for endothelial and smooth muscle cell suspensions and vascular strips in vitro do not exceed M(mt) . We conjecture that most of the reported values of intravascular O(2) flux are overestimated, and the likely source is in the experimental estimates of convective O(2) transport at upstream and downstream points of unbranched vascular segments. Planta, 2000 Jun, 211(1), 43 - 9 Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana; Huertas IE et al.; Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry . Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration . The magnitude of this release was dependent on the length of time the cells had been kept in the dark . Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells . External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport . Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) . The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark . Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3- . These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium. Endocr Regul, 2000 Jun, 34(2), 73 - 81 The influence of thyroxine on intensity of energy metabolism in bone marrow myeloid cells and neutrophilic polymorphonuclear leukocytes of neonatal pig; Babych H et al.; OBJECTIVES: To investigate the participation of thyroxine in the regulation of energy metabolism in neutrophilic polymorphonuclear leukocytes and their bone marrow precursors . MATERIALS AND METHODS: The influence of L-thyroxine (T4; 4 mg/kg every 12 hr from day 2 to 10 of age) was estimated on the activity of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), NADP-dependent isocitrate dehydrogenase (ICDH) and cytochrome C-oxidase in bone marrow myeloid cells and circulating neutrophils of 3, 5 and 10 day (d) old piglets . Serum T4 and 3,5, 3'-triiodothyronine (T3) concentrations were estimated at every stage of experiment by radioimmunoassay . Bone marrow cells of myeloid lineage and blood neutrophilic polymorphonuclear leukocytes were separated by differential centrifugation of haematopoietic cell suspension using Ficoll-Hypaque gradients . RESULTS: The hyperthyroid status resulted in significant increase in PFK and LDH activity in myelokaryocytes of 3 and 3-5 d piglets, while the activity of HK and PK in the cells of 3-10 d animals remained unchanged . Moreover, ICDH activity in myelokaryocytes increased on day 10 and that of cytochrome C oxidase in bone marrow cells at all intervals . Marked increase in HK and LDH activity on day 3-5 was found also in blood polymorphonuclear granulocytes, while PFK and PK activity was increased during the whole period . At the same time even the increase in ICDH and cytochrome C-oxidase activity was observed, respectively, in 3 and 5-10 d old piglet neutrophils . Besides that, T4 inhibited G-6-PDH activity in myeloid cells on day 3 to 10 and did not influence the enzyme activity in circulating leukocytes . CONCLUSIONS: The administration of T4 resulted in preferential stimulation of oxidative stages of carbohydrate catabolism in myelocaryocytes, while the activity of glycolytic enzymes in these cells was less affected . On the contrary, the enzymes of glycolysis in blood neutrophils showed higher sensitivity to T4 action as compared to catalysts of oxidative reactions . The intensity of pentose phosphate pathway seems to be inhibited in bone marrow myelocaryocytes of T4 treated animals, while that in blood leukocytes remained unaffected. Acta Physiol Scand, 2000 Jul, 169(3), 203 - 19 Ruled by waves? Intracellular and intercellular calcium signalling; Rottingen J et al.; The field of calcium signalling has evolved rapidly the last 20 years . Physiologists had worked with cytosolic Ca2+ as the coupler of excitation and contraction of muscles and as a secretory signal in exocrine glands and in the synapses of the brain for several decades before the discovery of cellular calcium as a second messenger . Development of powerful techniques for measuring the concentration of cytosolic free calcium ions in cell suspensions and later in single cells and even in different cellular compartments, has resulted in an upsurge in the knowledge of the cellular machinery involved in intracellular calcium signalling . However, the focus on intracellular mechanisms might have led this field of study away from physiology . During the last few years there is an increasing evidence for an important role of calcium also as an intercellular signal . Via gap junctions calcium is able to co-ordinate cell populations and even organs like the liver . Here we will give an overview of the general mechanisms of intracellular calcium signalling, and then review the recent data on intercellular calcium signals . A functional coupling of cells in different tissues and organs by the way of calcium might be an important mechanism for controlling and synchronizing physiological responses Int Arch Allergy Immunol, 2000 Jul, 122(3), 216 - 23 Inhibitory effect of wheat germ agglutinin on mouse mast cell adhesion to fibronectin; Wyczolkowska J et al.; BACKGROUND: Mast cells play a critical role in allergic and inflammatory responses . The interactions between these cells and extracellular matrix components influence the distribution of mast cells in tissues and their biological responsiveness . It has been reported that the lectin wheat germ agglutinin (WGA) inhibits mast cell mediator release . We decided to investigate whether adhesion to fibronectin (FN), another mast cell function, which is upregulated following FcepsilonRI cross-linking, is also inhibited by WGA . METHODS: Mouse bone-marrow-derived mast cell line MCP5/L was used . For FcepsilonRI-dependent mast cell activation, MCP5/L cells were sensitized with mouse IgE antibodies . WGA was added to cell suspensions simultaneously with a challenging agent and, after an appropriate incubation period, beta-hexosaminidase release and adhesion to FN were determined . RESULTS: Both FcepsilonRI cross-linking-dependent mast cell adhesion to FN and mediator release were dose-dependently inhibited by WGA; however, the lectin concentrations required to induce maximum inhibition of adhesion were significantly lower . Furthermore, WGA inhibited phorbol-myristate-acetate- and A-23187-mediated mast cell adhesion to FN, i.e . processes that do not engage FcepsilonRI . The effect of WGA on FcepsilonRI-mediated secretion was reversed by GlcNAc . In contrast, combination of GlcNAc and NeuNAc or N, N'-diacetylchitobiose was required to reverse the inhibitory effect of WGA on mast cell adhesion . CONCLUSION: The characteristics of WGA-mediated inhibition of MCP5/L mast cell adhesion to FN suggest that mast cell integrins are targets of the inhibitory action of WGA and the sugar moieties on these receptors might be important for receptor function . Biotechnol Bioeng, 2000 Sep 5, 69(5), 478 - 85 Hydrogen production by Anabaena variabilis PK84 under simulated outdoor conditions; Borodin VB et al.; Hydrogen production by autotrophic, vanadium-grown cells of Anabaena variabilis PK84, a cyanobacterial mutant impaired in the utilization of molecular hydrogen, has been studied under simulated outdoor conditions . The cyanobacterium was cultivated in an automated helical tubular photobioreactor (4.35 L) under air containing 2% CO(2), with alternating 12-h light (36 degrees C) and 12-h dark (14 degrees to 30 degrees C) periods . A . variabilis steadily produced H(2) directly in the photobioreactor during continuous cultivation for 2.5 months . The maximum H(2) production by the continuously aerated culture under light of 332 microE . s(-1) . m(-2) was 230 mL per 12-h light period per photobioreactor and was observed at a growth density corresponding to 3.6 to 4.6 microgram Chl a . mL(-1) (1.2 to 1.6 mg dry weight . mL(-1)) . Replacement of air with an argon atmosphere enhanced H(2) evolution by a factor of 2 . This stimulatory effect was caused mainly by N(2) deprivation in the cell suspension . A short-term decrease of the CO(2) concentration in the air suppressed H(2) evolution . Anoxygenic conditions over the dark periods had a negative effect on H(2) production . The peculiarity of hydrogen production and some physiological characteristics of A . variabilis PK84 during cultivation in the photobioreactor under a light-dark regime are investigated . Tohoku J Exp Med, 2000 May, 191(1), 7 - 20 Auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration: short-term results; Abe T et al.; Autologous iris pigment epithelial cell transplantation was performed on patients with exudative age-related macular degeneration (AMD) . Autologous IPE cell culture was performed using autologous serum after iridectomy in 7 patients with AMD . The cell suspensions (2 approximately 20 x 10(4) cells) were transplanted into the submacular lesion of individuals after removal of neovascular membranes . Subsequent ophthalmological examinations, including best corrected visual acuity and fluorescein or indocyanine green angiography, were performed . In addition, 15 patients with AMD, who underwent removal of neovascular membrane without transplantation, were evaluated as non randomized controls . Varying degrees of atrophy or defects of choriocapillaris and retinal pigment epithelium were observed in all of the patients . No cystoid macular edema or fluorescein leakage was observed after treatment, but window defects were present . No patient had decreased visual acuity . One treated patient developed mild subretinal fibrosis and an other patient developed mild preretinal fibrosis, however no difference was significant when compared with the control . In conclusion, the treatment resulted in no significant improvement in macular function, as compared with the control; however, no rejection or deterioration in visual acuity occurred up to the 13 month follow up. Magn Reson Med, 2000 Jul, 44(1), 144 - 56 NMR relaxation in tissues with weak magnetic inhomogeneities; Jensen JH et al.; A theory is presented for describing the effect on the transverse NMR relaxation rate of microscopic spatial inhomogeneities in the static magnetic field . The theory applies when the inhomogeneities are weak in magnitude and the nuclear spins diffuse a significant distance in comparison with a length scale characterizing the inhomogeneities . It is shown that the relaxation rate is determined by a temporal correlation function and depends quadratically on the magnitude of the inhomogeneities . For the case of unrestricted diffusion, a simple algebraic approximation for the temporal correlation function is derived . The theory is illustrated by applying it to a model of randomly distributed magnetized spheres . The theory is also used to fit experimental data for the dependence of the relaxation rate on the interecho time for a Carr-Purcell-Meiboom-Gill pulse sequence . The experimental systems considered are in vitro red blood cell suspensions and samples of human gray matter and rat liver . Magn Reson Med 44:144-156, 2000 . Plant Physiol, 2000 Jul, 123(3), 1029 - 36 Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins; Inouhe M et al.; Suspension-cultured cells of azuki bean (Vigna angularis) as well as the original root tissues were hypersensitive to Cd (<10 microM) . Repeated subculturings with a sublethal level of Cd (1-10 microM) did not affect the subsequent response of cells to inhibitory levels of Cd (10-100 microM) . The azuki bean cells challenged to Cd did not contain phytochelatin (PC) peptides, unlike tomato (Lycopersicon esculentum) cells that have a substantial tolerance to Cd (>100 microM) . Both of the cell suspensions contained a similar level of reduced glutathione (GSH) when grown in the absence of Cd . Externally applied GSH to azuki bean cells recovered neither Cd tolerance nor PC synthesis of the cells . Furthermore, enzyme assays in vitro revealed that the protein extracts of azuki bean cells had no activity converting GSH to PCs, unlike tomato . These results suggest that azuki bean cells are lacking in the PC synthase activity per se, hence being Cd hypersensitive . We concluded that the PC synthase has an important role in Cd tolerance of suspension-cultured cells. Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 99 - 102 {Effects of media on the production of flavonoids by suspension cultures of Saussurea medusa}; Zhao DX et al.; Flavonoids were produced from cell suspension cultures of Saussurea medusa . The results of studies on eight types of culture media showed that the MS medium was the best for cell growth and flavonoids formation, We investigated the effects of all the components of MS medium on the cell growth and flavonoids production, and found that carbon, nitrogen and phytohormone had especially marked effects . With MG medium a modified MS medium, the yield of cell growth was 24.8 g(dwt)/L, with MP medium another modified MS mediums, the yield of flavonoids production was 1 . 75 g/L . The yield of cell growth and flavonoids production in MG and MP medium were 32% and 70% higher than that in MS medium respectively. Gen Comp Endocrinol, 2000 Jul, 119(1), 95 - 104 Effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail Coturnix coturnix japonica; Moore CB et al.; Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail . The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail . The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL) . In the second experiment, melatonin was administered in varying doses to adult quail placed in LL . The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 microg/ml melatonin given in the drinking water for 16 h per day for 2 weeks . The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment . In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response . To evaluate the humoral immune response, primary antibody titers were calculated 7 days postintravenous injection with a Chukar red blood