Protein Expr Purif, 2000 Jun, 19(1), 131 - 8 Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells; James EA et al.; Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions . The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator . In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene . For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA . Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls . Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF . Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium . The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional . The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells . The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels . Microbiology, 2000 May, 146 ( Pt 5), 1091 - 8 High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp . strain HE5; Schrader T et al.; Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil . The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis . An identity of 99.4% was obtained to Mycobacterium gilvum; however, the type strain was unable to utilize morpholine . A maximal growth rate of 0.17 h(-1) was observed at a morpholine concentration of 30 mM, 30 degrees C and pH 7.2 . The substrate was tolerated at concentrations up to 100 mM . Besides morpholine, the strain utilized pyrrolidine, piperidine and proposed intermediates in morpholine metabolism such as glycolate, glyoxylate and ethanolamine . Degradation of morpholine, piperidine and pyrrolidine by resting or permeabilized cells was strictly dependent on the presence of oxygen . Addition of the cytochrome-P450-specific inhibitor metyrapone to the growth medium resulted in a significantly decreased growth rate if these cyclic amines were used as a substrate . Carbon monoxide difference spectra of crude extracts from cells grown on these substrates compared to spectra obtained for extracts of succinate-grown cells indicated that cytochrome P450 is specifically expressed during growth on the cyclic amines . These data indicated that a cytochrome-P450-dependent monooxygenase is involved in the degradation of the three cyclic amines. Ann Occup Hyg, 2000 Jun, 44(4), 259 - 69 Inflammatory potential of dust from waste handling facilities measured as IL-8 secretion from lung epithelial cells in vitro; Allermann L et al.; OBJECTIVES: Organic dust contains several different components which may cause pulmonary effects, and many health problems have been associated with the collection and recycling of organic waste . It is often difficult to obtain a precise measurement of the exposure to each component in dust, and organic dust samples obtained from different workplaces may vary profoundly in composition . The aim of this study was to evaluate the inflammatory potential of dust from different waste handling plants . Furthermore, we set out to investigate the role of endotoxin in the inflammatory potential of dust . METHODS: Dust samples were obtained from four incineration plants, three samples from a plant sorting household waste, five paper-sorting plants, two mail centres, four bottle-sorting plants, and two combined paper-sorting and composting plants . The samples were tested in a bioassay with the lung epithelial cell line A549 . Cells were stimulated for 24h with dust samples at six concentrations, and subsequently the interleukin 8 (IL-8) secretion into the growth medium was measured . The initial slope of the dose response curves was used to calculate the potency factor (PF) of the dust samples, and correction against positive control samples was used to reduce day-to-day variation . The concentration of endotoxin in the dust samples was measured by the limulus amebocyte lysate (LAL) assay . RESULTS: The inflammatory potential of the dust samples for dust from the paper- and mail-sorting plants showed a significantly lower PF as compared with dust from the plants handling mixed household waste . A significantly lower PF for the dust samples from the bottle-sorting plants (excluding one outlier plant) compared with dust from the plants handling mixed household waste was also found . No correlation was observed between the PF and the concentration of endotoxin in the samples . CONCLUSION: The PFs obtained seem to reflect the material handled, with mixed household waste generating organic dust with the highest inflammatory potentials. Biometals, 2000 Mar, 13(1), 73 - 6 Kinetics of SO4(-2) reduction under different growth media by sulfate reducing bacteria; Mohanty SS et al.; Sulfate Reducing Bacteria (SRB) were used to reduce the SO4(-2) concentration in waste water . The growth pattern of SRB was found by varying the concentration of nutrients and the biomass . The specific reaction constant was evaluated in each case. Electrophoresis, 2000 Apr, 21(7), 1372 - 80 Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures; Jensen PK et al.; Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection . This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization . We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization . Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E . coli) and Deinococcus radiodurans (D . radiodurans) . We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression . The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes. J Cell Physiol, 2000 Jul, 184(1), 17 - 26 Cell-cycle-dependent turnover of P-glycoprotein in multidrug-resistant cells; Zhang W et al.; Regulation of P-glycoprotein (Pgp) expression occurs not only at the DNA and mRNA level but also at the protein level . We showed previously that Pgp was stabilized when multidrug-resistant CH(R)C5 and SKVCR 2.0 ovarian cell lines were subjected to serum-starved or high-cell-density growth conditions, whereas Pgp turnover in a leukemic multidrug-resistant cell line, CEMVLB0.1, was not affected by serum starvation (Muller et al., 1995) . On further analysis, we have observed that the majority of the CH(R)C5 and SKVCR 2.0 cells under these conditions were in the G1/G0 phase of the cell cycle, whereas the cell cycle of CEMVLB0.1 cells was not affected . Pgp in CEMVLB0.1 cells was stabilized only when the cell cycle was delayed in the G1/G0 phase by using amino acid-deficient growth medium . In CH(R)C5 cells, Pgp half-life was also considerably increased when the cell cycle of these ovary-derived cells was delayed in the G1/G0 phase by using high concentrations of progesterone under normal serum growth conditions . In contrast, Pgp stability was not greatly affected if these cells were delayed in the S or G2/M phase of the cell cycle with Ara-C, cisplatin, or colchicine under the same conditions . Insulin-like growth factors could release the serum-starved CH(R)C5 and SKVCR2.0 cells from the G1/G0 phase and destabilized Pgp . These results indicate that Pgp turnover is a cell-cycle-related process in MDR cells . J Nucl Med Technol, 2000 Jun, 28(2), 94 - 5 Is your technetium generator eluate sterile? Snowdon GM. OBJECTIVE: This study was performed to assess the sterility of multidose 99mTc generator eluate vials at the end of a working day . METHODS: Expired 99mTc generator eluate vials were collected over a period of 10 wk and stored until the activity reached background . Four batches of 10 vials each were selected randomly and sent to an independent microbiology laboratory for sterility testing . RESULTS: No eluate showed any microbial growth after 14 d incubation in growth media . CONCLUSION: Retrospective sterility testing of 99mTc generator eluate confirmed the validity of our departmental protocol for radiopharmaceutical preparation . Sterility testing has become part of our quality control program. J Agric Food Chem, 2000 May, 48(5), 1537 - 41 Purification and kinetic characterization of an anionic peroxidase from melon (Cucumis melo L.) cultivated under different salinity conditions; Rodriguez-Lopez JN et al.; The partial characterization of an anionic peroxidase in melon fruit is described . Four melon peroxidase (MPX) isoenzymes were detected in crude extracts after isoelectric focusing . The major MPX isoenzyme (pI = 3.7) was partially purified by including hydrophobic and anion-exchange chromatography in the purification scheme . The sample obtained was used to characterize MPX . This peroxidase did not show activity on ascorbic acid but oxidized guaiacol at a high rate, showing an optimum pH of 5.5 when acting on this last reducing substrate . Melon fruits grown under highly saline conditions showed slightly increased levels of this anionic isoenzyme . Kinetic studies using 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) as reducing substrate showed that increased salinity in the growth medium did not modify the kinetic parameters of melon peroxidase on both hydrogen peroxide and reducing substrate. J Surg Res, 2000 Jun 1, 91(1), 26 - 31 The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing; Berger AC et al.; BACKGROUND: Endostatin is a potent angiogenesis inhibitor, which is currently being used in Phase I trials as an antitumor agent . The purpose of this study was to determine whether endostatin has an effect on wound healing in a murine model . MATERIALS AND METHODS: The function of endostatin was confirmed using a human microvascular endothelial cell (HMVEC) proliferation assay in which cells are treated for 4 days with growth media plus or minus endostatin . Full-thickness incisions were made on the dorsum of athymic nude mice and closed primarily with skin staples . PVA sponges were implanted in some wounds to determine vascular ingrowth . Subsequently, mice were treated with recombinant human endostatin at 20 mg/kg/day or 50 mg/kg/dose BID versus control for a total of 14 days . On Days 2, 4, 8, 12, and 16, three mice per group had serum samples drawn and were sacrificed . Perpendicular breaking strength (N) was determined using an Instron 5540 tensometer . Wound strength was determined by dividing breaking strength by wound area (N/cm(2)) . Vascular density in sponges was determined using CD31 immunohistochemistry . Serum endostatin concentrations were determined using a commercially available ELISA kit . RESULTS: Endostatin caused a significant reduction of endothelial cell proliferation after 4 days compared to media alone (72%, P = 0.031) . At all time points tested, there was no statistical difference in the wound-breaking strength between endostatin and control-treated mice at either the low or high dose . Serum endostatin levels were consistently 10-fold higher in endostatin-treated mice than in controls . No differences in vascular density were seen in endostatin versus control-treated mice as determined by CD31 immunohistochemistry of PVA sponges . CONCLUSION: Therapy with human endostatin does not induce a significant decrease in breaking strength of cutaneous wounds in mice. Arch Microbiol, 2000 Apr, 173(4), 245 - 52 Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae; Slaninova I et al.; Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate . During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed . The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose . The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls . Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface . The rate of incorporation of D-{U-14C}glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics . No differences in cell wall composition between osmotically stressed yeast and control cells were found . Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments . After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds . The response of S . cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain . We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis. Cell Biol Toxicol, 1999, 15(5), 299 - 309 Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus; Ganassin RC et al.; A cell line, PHL, has been successfully established from newly hatched herring larvae . The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing . These cells retain a diploid karotype after 65 population doublings . PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant . Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills . In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products . This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring . In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species. Cell Biol Toxicol, 1999, 15(5), 291 - 7 The SOS induction of umu'-'lacZ fusion gene by oxidative damage is influenced by polyamines in Escherichia coli; Oh TJ et al.; We report that polyamines have an effect on the SOS response of the umu operon in polyamine-deficient mutant and wild-type Escherichia coli strains carrying the umu'-'lacZ fusion . H2O2 effectively induces umu'-'lacZ in the wild type, but not significantly in the mutant strain . Exogenous polyamines did not restore the umu induction in the mutant to the wild-type level . In logarithmically growing cells, the basal expression of umu gene in the mutant is about five times higher than that of the wild type . The addition of polyamines to the growth medium markedly reduces the basal expression to the wild-type level . This reduction is due not to growth rate but to the polyamine itself . Our results suggest that polyamines are essentially involved in the SOS induction of the umu operon in E . coli. Mikrobiologiia, 2000 Jan-Feb, 69(1), 13 - 8 {Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions}; Berg IA et al.; The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied . Both in the light and in the dark, acetate assimilation in Rsp . rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium . The assimilation of propionate was accompanied by the excretion of pyruvate . Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate . These data implied that the citramalate (CM) cycle in Rsp . rubrum cells grown aerobically in the dark can function as an anaplerotic pathway . This supposition was confirmed by respiration measurements . The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium . The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway . The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle . The addition of 5 mM itaconate to extracts of Rsp . rubrum cells inhibited the activity of this enzyme by 85% . The data obtained suggest that the CM cycle continues to function in Rsp . rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically. Curr Genet, 2000 Apr, 37(4), 268 - 75 Copper-dependence of mitochondrial DNA rearrangements in Podospora anserina; Borghouts C et al.; Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P . anserina . These rearrangements include the systematic amplification of the first intron (p1-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (p1DNA) . In addition, deletions and amplifications of other regions of the mtDNA occur . The molecular basis of the underlying processes is not understood in detail . A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper . In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the p1-intron and the transposition of this mobile element, are not impaired . In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of p1DNA, appear to be affected . Additional copper in the growth medium rescues this molecular phenotype . We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements. Biotechnol Bioeng, 2000 Jun 20, 68(6), 619 - 27 Two-phase model of the kinetics of growth of Rhizopus oligosporus in membrane culture; Ikasari L et al.; An empirical model was developed to describe a growth profile occurring in solid-state fermentation (SSF), namely that consisting of an initial period of rapid acceleration followed by an extended period of deceleration . This kinetic profile is not adequately described by the logistic model . The empirical model is based on the concept of active and nonactive hyphal segments . Exponential and deceleration growth phases are modeled . The model parameters can be determined directly from the dry-weight profile and they depend on the growth medium present . The model suggests that, at the instant the culture enters the deceleration phase, there is a 71% to 86% decrease in the number of actively extending hyphal tips and that, during the deceleration phase, there is an exponential decay in the number of active hyphal segments, with a first-order decay constant of 0.042 to 0.072 h(-1) . J Surg Res, 2000 May 15, 90(2), 113 - 8 Neuroblastoma and hepatocyte coculture conditioned media alter apoptosis; Beierle EA et al.; BACKGROUND: Neuroblastoma is a childhood tumor that often displays unusual biological behavior . The tumor may present with widespread metastases that are unresponsive to aggressive treatment . At other times, both the metastases and the primary tumor may spontaneously regress without treatment . Apoptosis, or programmed cell death, is thought to play a role in the dichotomous behavior of neuroblastoma . We hypothesize that neuroblastoma cells will interact with host tissues to release mediators that affect apoptosis . MATERIALS AND METHODS: Human neuroblastoma cells and human Chang hepatocytes are grown in a noncontact, coculture system . After incubation for 4 days, the medium from the coculture system is collected . Neuroblastoma cells and Chang hepatocytes are then plated separately with the conditioned medium and their own standard growth medium as controls . After 4 days, these cells are harvested and cytospins made for immunostaining . Tumor necrosis factor alpha (TNF-alpha), Fas ligand, and Bcl-2, are measured with immunohistochemistry . Apoptosis is detected with the TUNEL method . Immunostaining data are interpreted with computer image analysis and reported as stain index . TUNEL data are reported as percentage apoptotic cells . All data are reported as means +/- SEM . Statistical analysis is performed and P < 0.05 considered significant . RESULTS: Chang hepatocytes grown in the coculture conditioned media have an increase in TNF-alpha and Fas ligand . The neuroblastoma cells have a significant decrease in Fas ligand . There is a significant increase in the number of apoptotic hepatocytes when they are cultured in the conditioned media . In contrast, the neuroblastoma cells grown in the coculture conditioned media show no increase in apoptosis . Finally, Bcl-2 is significantly increased in the neuroblastoma cells cultured in the conditioned media . CONCLUSIONS: Neuroblastoma cells grown in coculture conditioned media show increased expression of Bcl-2 and decreased Fas ligand levels . These changes should diminish apoptosis activity in the tumor cells . In contrast, the conditioned media induce elevated levels of proapoptotic mediators in the Chang hepatocytes . A tumor's ability to successfully metastasize may be dependent on mediators generated in the tumor-host interaction, and may not be just an independent characteristic of the tumor itself . Mol Microbiol, 2000 Apr, 36(2), 261 - 77 Two novel proteins, PopB, which has functional nuclear localization signals, and PopC, which has a large leucine-rich repeat domain, are secreted through the hrp-secretion apparatus of Ralstonia solanacearum; Gueneron M et al.; The Ralstonia solanacearum hrp gene cluster codes for components of a type III secretion pathway necessary for the secretion of PopA1, a hypersensitive response-like elicitor protein . In the present study, we show that several other Hrp-secreted proteins can be detected by growing wild-type bacteria in minimal medium in the presence of Congo red . Two of these proteins, PopB and PopC, are encoded by genes located downstream of popA and constitute an operon with popA . popABC mutants retain the wild-type ability to cause disease in hosts and to elicit the hypersensitive response on non-hosts . Expression of the popABC operon is controlled by the hrpB regulatory gene and is induced upon co-culture with Arabidopsis cell suspensions . This plant cell-specific induction depends on PrhA, a putative receptor for plant specific signal(s) . The transcription of the popABC operon is not modified by the addition of Congo red to the growth medium and the intracellular pools of PopB and PopC are very similar in the absence or presence of Congo red . Preliminary data suggest that Congo red stabilizes secreted proteins in the extracellular medium . PopB is a 173-amino-acid-basic protein that contains a functional bipartite nuclear localization signal . PopC is a 1024-amino-acid protein that carries 22 tandem leucine-rich repeats (LRR) . The LRR domain of this protein forms a consensus that perfectly matches the predicted eukaryotic cytoplasmic LRR consensus . We propose that PopB and PopC may be translocated into plant cells via the Hrp pathway. Genes Cells, 2000 Apr, 5(4), 239 - 50 Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti-terminator BglG, CRP-cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiation; Gulati A et al.; BACKGROUND: Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic beta-glucosides, is extremely sensitive to the presence of glucose in the growth medium . We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression . RESULTS: Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose . Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho-independent terminator present within the leader sequence . Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose-mediated repression is the anti-terminator protein, BglG . Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression . Studies using crp, cya and crr strains show that both CRP-cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation . Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator . Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose-grown cultures of a crp+ strain . Glucose repression is lost in the crr mutant strain . CONCLUSIONS: The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti-termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF . The CRP-cAMP complex is also involved in this regulation . The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes. Arch Physiol Biochem, 1999 Oct, 107(4), 286 - 91 Luteinizing hormone releasing hormone increases proliferation of meningioma cells in vitro; Durmaz R et al.; The fact that meningioma shows at least a 2:1 predilection for women over men is considered to be due to endocrinological and paracrine regulation of the development of this tumour . The presence of receptors for the luteinizing hormone releasing hormone (LHRH) in gynaecological cancer permits the use of LHRH agonistic or antagonistic analogues with a direct effect or by the gonado-pituitary axis suppression in the treatment of these tumours . Therefore, the effect of LHRH on meningioma cells is tested in this study . Meningioma cells from three female patients were cultured and LHRH (50 ng/ml) was added to the growth medium daily, for fourteen days . At the end of this period the cells were counted by means of a Coulter Counter . The stimulating effects of LHRH on the increase of the amount of cells in the meningioma monolayer culture were 146% (p < 0.01), 134% (p < 0.05) and 141% (p < 0.05) of the control, respectively, for the three patients. In Vitro Cell Dev Biol Anim, 2000 Mar, 36(3), 194 - 200 Quantitative assessment of marine sponge cells in vitro: development of improved growth medium; Willoughby R et al.; As sources of natural products with potential human therapeutic value, marine sponges are important subjects for cell culture studies . A critical component of any cell culture system is its growth medium . Proceeding from the hypotheses that the thawed, cryopreserved, primary cells would display detectable differential responses and that those responses could be comparatively quantified, this study has established that multiwell screening assays are useful tools for improving medium formulations in cell cultures of the marine sponge, Teichaxinella morchella . Fluorescent probe signals were correlated with known cell densities and viabilities in a 96-well format . Analysis of variance and post-test methods were applied to judge the significance of signal differences seen in a variety of medium formulations . Results from a series of experiments suggested that reducing glutamine and selenium concentrations in the standard medium would result in greater DNA, protein, and esterase activity signals . This was confirmed by the direct comparison of the standard and improved medium formulations . Significantly higher protein content and esterase activity were associated with the improved medium . DNA content was also higher, though not significantly . The result is a new medium formulation that may be more able to support cell growth and division, providing an improved cell culture system for marine sponge cell studies . The assays can be used in additional studies to further improve the in vitro conditions for marine sponge cell culture. Microbiol Res, 2000 Mar, 154(4), 339 - 47 Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.); Omar SA et al.; The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi . During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt) . The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi . Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately . All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates . Four fungal species, Aspergillus awamori, A . flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation . The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion . Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity . Cu(1+), Fe3+ and Al3+ showed a stimulatory effect . Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities . Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%. Eur J Pharm Sci, 2000 May, 10(3), 187 - 93 A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides; Jaaskelainen I et al.; Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA . To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared . The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer . Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene . Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method . Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol) . Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL . In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1) . The polymeric systems were ineffective . The effect of the complexation medium was further studied on CV-1 cells . Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM) . Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size . Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes . In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides. Rev Bras Biol, 1999 Aug, 59(3), 517 - 25 Mitogenic activity of fetal bovine serum, fish fry extract, insulin-like growth factor-I, and fibroblast growth factor on brown bullhead catfish cells--BB line; Cyrino JE et al.; Bioassays were performed to assess the effects of different levels of growth medium supplementation with fetal bovine serum (FBS), fish fry extract (FE), combinations of FBS and FE, and addition of insulin-like growth factor I (IGF-I) and fibroblast growth factor (FGF) on the proliferation of brown bullhead catfish cells (BB line) . Treatments (n = 4) were: 2.5, 5, 10, and 15.0% FBS or FE and 5/2.5, 5/5, 10/2.5, and 10/5 of a FBS/FE combination as supplement to the growth medium, or the addition of 0.1, 1, 2.5, 10, 25, and 75 ng/ml of either IGF-I or FGF to the growth media . Initial cell density was 1.1 x 10(6) cells per well on uncoated 24-well plates . Incubation temperature was 29.5 +/- 0.7 degrees C . Six hours after plating, initial culture medium was removed, plates rinsed with Dulbecco's phosphate buffered saline, treatment media added, and cells allowed to proliferate for 24 hours . Another bioassay was performed with rat myoblast omega cells (RMo) using the same levels of growth medium supplemented with FBS, FE and FBS/FE . Base growth medium was Dulbecco's MEM . The initial cell density was 7.2 x 10(6) cells per well, and the bioassay was carried out at 36.0 +/- 0.5 degrees C, on a 95% air, 5% CO2 incubator . Increasing levels of FBS had a positive effect (P < 0.05) on the proliferation of both BB and RMo cells . Increasing levels of FE had a negative effect (P < 0.05) on the proliferation of BB cells and totally inhibited the proliferation of RMo cells at any level of supplementation . Higher levels of FE on the FBS/FE combinations presented a negative effect on the proliferation of both BB and RMo cells (P < 0.05) . Insulin-like growth factor I had a positive quadratic effect (P < 0.05) on the proliferation of BB cells . Apparently, mammalian growth factors slightly stimulated mitogenic activity in fish cells, while FE contained factors which inhibited the mitogenic activity of RMo and BB cell lines. Anal Chem, 2000 Apr 1, 72(7), 1666 - 71 UV resonance Raman detection and quantitation of domoic acid in phytoplankton; Wu Q et al.; Cultures of the phytoplankton diatom, Pseudonitzschia multiseries, have been harvested under controlled growth conditions ranging from late logarithmic to late stationary phase (17-58 days) . The amount of domoic acid (DA) present in the growth media and in the homogenized cells has been determined by HPLC . Defined samples of media, homogenized cells, whole cells, and whole cells in media have been laser excited at 251 nm for the purpose of selectively exciting intense UV resonance Raman spectra from DA in the samples . Neither media nor cell component spectra from algae seriously interfere with DA spectra . The spectral cross sections for the dominant 1652-cm-1 mode of DA have been determined for 242-, 251-, and 257-nm excitation . Maximum sensitivities are achieved with 251-nm excitation because cross sections for DA are a maximum, and interference from other algal components becomes very small . DA concentrations that have been determined with 251-nm excitation by resonance Raman methods correlate closely with values determined independently with HPLC, especially at higher DA concentrations . The UV resonance Raman analysis of DA in phytoplankton algae is shown to be very sensitive and quantitative as well as rapid and nonintrusive. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 37 - 48 Glucose starvation induces a drastic reduction in the rates of both transcription and degradation of mRNA in yeast; Jona G et al.; Gradual depletion of essential nutrients in yeast cultures induces a complex physiological response, leading initially to induction of pathways required for the utilization of alternative nutrients and, when such sources are depleted, to entry into stationary phase . Abrupt removal of sugar does not allow the proper establishment of stationary phase . Here we report that abrupt removal of glucose from the growth medium elicits a coordinated response in yeast cells that resembles, in some aspects, the gradual passage to stationary phase . Phosphorylation of RNA polymerase II at a subset of sites in the COOH-terminal domain (CTD) is decreased . Transcription by RNA polymerases I and II is shut down almost completely, whereas transcription by RNA polymerase III continues . In parallel, the rate of mRNA degradation is drastically reduced, at a stage preceding poly(A) shortening . This response is suited for conservation of scarce resources while preserving the ability of cells to recover when nutrients become available. Eur J Biochem, 2000 Apr, 267(8), 2334 - 9 Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent; Sumper M et al.; A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures . In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion . At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis . Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells . Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family . Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state . Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors. Plant Physiol, 2000 Apr, 122(4), 1387 - 97 Enhancement of Na(+) uptake currents, time-dependent inward-rectifying K(+) channel currents, and K(+) channel transcripts by K(+) starvation in wheat root cells; Buschmann PH et al.; Excessive low-affinity Na(+) uptake is toxic to the growth of glycophytic plants . Recently, several reports have suggested that the interaction between K(+) and Na(+) uptake might represent a key factor in determining the Na(+) tolerance of plants . We investigated the effects of K(+) starvation on Na(+) and K(+) uptake mechanisms in the plasma membrane of wheat (Triticum aestivum L.) root cortex cells using the patch-clamp technique . Unexpectedly, K(+) starvation of wheat seedlings was found to enhance the magnitude and frequency of occurrence of time-dependent inward-rectifying K(+) channel currents (I(K)(+)(in)) . We examined whether the transcription of a wheat root K(+)(in) channel gene is induced by K(+) starvation . A cDNA coding for a wheat root K(+) channel homolog, TaAKT1 (accession no . AF207745), was isolated . TaAKT1 mRNA levels were up-regulated in roots in response to withdrawal of K(+) from the growth medium . Furthermore, K(+) starvation caused an enhancement of instantaneous Na(+) currents (I(Na)(+)) . Electrophysiological analyses suggested that I(K)(+)(in) and I(Na)(+) are not mediated by the same transport protein based on: (a) different activation curves, (b) different time dependencies, (c) different sensitivities to external Ca(2+), and (d) different cation selectivities . These data implicate a role for I(Na)(+) in Na(+) uptake and stress during K(+) starvation, and indicate that K(+)(in) channels may contribute to K(+)-starvation-induced K(+) uptake in wheat roots. Genetics, 2000 Mar, 154(3), 1085 - 99 DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae; Moore CW et al.; Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination . Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains . DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium) . Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis . After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions {liquid holding (LH)} . Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains . After low doses, chromosomes were sometimes degraded and reconstructed during LH . Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH . These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S . cerevisiae when homologous recombination is impaired. Development, 2000 May, 127(9), 1961 - 9 The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by alphaV integrins; Blaschuk KL et al.; We have previously shown that oligodendrocyte progenitor cells exhibit developmental switching between alphav-associated beta integrin subunits to sequentially express alphavbeta1, alphavbeta3 and alphavbeta5 integrins during differentiation in vitro . To understand the role that alphavveta3 integrin may play in regulating oligodendrocyte progenitor cell behaviour, cells of the rat cell line, CG-4, were genetically engineered to constitutively express alphavbeta3 integrin by transfection with full-length human beta3 integrin subunit cDNA . Time-lapse videomicroscopy showed no effect of beta3 expression on cell migration but revealed enhanced proliferation on vitronectin substrata . Comparison of mitotic indices, as measured by 5-bromo-2'-deoxyuridine incorporation, confirmed that human beta3 integrin-expressing cells exhibited enhanced proliferation, as compared to both vector-only transfected, and wild-type CG-4 cells when switched to differentiation medium from growth medium, but only in cultures grown on vitronectin and not on poly-D-lysine . The effects on proliferation were inhibited by a function-blocking antibody specifically directed against the human beta3 integrin subunit . Human beta3 integrin-expressing cells also exhibited reduced differentiation . This differentiation could be reduced still further by a function-blocking monoclonal antibody against alphavbeta5 integrin, as could differentiation in the wild-type CG-4 cells . Taken together, these results suggest that alphavbeta3 integrin may regulate oligodendroglial cell proliferation and that both downregulation of alphavbeta3 integrin expression and signalling through alphavbeta5 integrin may be critical to continued differentiation in vitro. J Biol Chem, 2000 May 26, 275(21), 16227 - 34 The aconitase function of iron regulatory protein 1 . Genetic studies in yeast implicate its role in iron-mediated redox regulation; Narahari J et al.; Iron regulatory proteins (IRP) are sequence-specific RNA-binding proteins that mediate iron-responsive gene regulation in animals . IRP1 is also the cytosolic isoform of aconitase (c-aconitase) . This latter activity could complement a mitochondrial aconitase mutation (aco1) in Saccharomyces cerevisiae to restore glutamate prototrophy . In yeast, the c-aconitase activity of IRP1 was responsive to iron availability in the growth medium . Although IRP1 expression rescued aco1 yeast from glutamate auxotrophy, cells remained growth-limited by glutamate, displaying a slow-growth phenotype on glutamate-free media . Second site mutations conferring enhanced cytosolic aconitase-dependent (ECA) growth were recovered . Relative c-aconitase activity was increased in extracts of strains harboring these mutations . One of the ECA mutations was found to be in the gene encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDP2) . This mutation, an insertion of a Ty delta element into the 5' region of IDP2, markedly elevates expression of Idp2p in glucose media . Our results demonstrate the physiological significance of the aconitase activity of IRP1 and provide insight into the role of c-aconitase with respect to iron and cytoplasmic redox regulation. J Appl Microbiol, 2000 Mar, 88(3), 388 - 403 The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts; Boeira LS et al.; The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined . Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods . The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth . When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls . However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture . As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied. Biochem J, 2000 Apr 1, 347 Pt 1, 37 - 44 Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions; Sheehan JK et al.; We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation . The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin . Similar antisera raised against the MUC2 and MUC5B mucins were not reactive . The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis . The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm) . Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands . Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated . M(r) measurements showed that these bands differed by single monomer units . The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments . The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo . In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro. Mol Cell Endocrinol, 2000 Feb 25, 160(1-2), 67 - 73 Differential transcriptional activation of peroxisome proliferator-activated receptor gamma by omega-3 and omega-6 fatty acids in MCF-7 cells; Thoennes SR et al.; While the role of dietary fats in breast cancer remains controversial, the recent cloning of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor, from human breast cancer cells lines provides a potential molecular link . Several fatty acids from four classes of dietary fats were tested for their ability to mediate the transcriptional activity of PPARgamma in MCF-7 and MDA-MB-231 cells using growth media with minimal serum . Whereas omega-3 fatty acids inhibit transactivation of PPARgamma to levels below control, omega-6, monounsaturated and saturated fatty acids stimulate the activity of the transcriptional reporter . These studies indicate that individual fatty acids differentially regulate the transcriptional activity of PPARgamma by selectively acting as agonists or antagonists . Furthermore, the transcriptional activation of PPARgamma correlates with cell proliferation in MCF-7 cells . Understanding the effects of individual fats on breast cancer cells and PPARgamma transactivation could provide important new insights into the epidemiology of breast cancer and the role of dietary fat. J Biol Chem, 2000 Mar 17, 275(11), 7597 - 603 Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity; Gable K et al.; Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine . The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the alpha-oxoamine synthase family required for serine palmitoyltransferase activity . The other alpha-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p . Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae . S . cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or phytosphingosine in the growth medium . The tsc3 mutant cells have severely reduced serine palmitoyltransferase activity . The TSC3 gene encodes a novel 80-amino acid protein with a predominantly hydrophilic amino-terminal half and a hydrophobic carboxyl terminus that is membrane-associated . Tsc3p coimmunoprecipitates with Lcb1p and/or Lcb2p but does not bind as tightly as Lcb1p and Lcb2p bind to each other . Lcb1p and Lcb2p remain tightly associated with each other and localize to the membrane in cells lacking Tsc3p . However, Lcb2p is unstable in cells lacking Lcb1p and vice versa. Plant Physiol, 2000 Mar, 122(3), 731 - 6 Differential expression of photosynthesis and nitrogen fixation genes in the cyanobacterium Plectonema boryanum; Misra HS et al.; The filamentous non-heterocystous cyanobacterium Plectonema boryanum fixes dinitrogen at a high rate during microaerobic growth in continuous illumination by temporal separation of oxygen-evolving photosynthesis and oxygen-sensitive dinitrogen fixation . The onset of nitrogen fixation is preceded by a depression in photosynthesis that establishes a sufficiently low level of dissolved oxygen in the growth medium . A several-fold reduction in the level of transcripts coding for phycocyanin (cpcBA) and the chlorophyll a binding protein of photosystem II (psbC) and psbA accompanied the depression in photosynthetic oxygen evolution . Unlike most of the other organisms examined to date, in P . boryanum, psbC and psbD do not appear to be co-transcribed . The psbC transcripts were down-regulated several fold, while the psbD transcript declined marginally during the nitrogen fixation phase . A decrease in dissolved oxygen and a dramatic increase in the level of nifH transcripts and the enzyme activity of nitrogenase were characteristic of the nitrogen fixation phase . The level of transcript for glnA, which encodes glutamine synthetase, was not altered . Reciprocal regulation of gene expression was well orchestrated with the alternating cycles of photosynthesis and nitrogen fixation in P . boryanum. Plant Physiol, 2000 Mar, 122(3), 705 - 14 Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation; Bernhardt C et al.; The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types . We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings . Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development . AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation . In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium . In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings . These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs. Mol Biol Cell, 2000 Mar, 11(3), 833 - 48 Glucose depletion rapidly inhibits translation initiation in yeast; Ashe MP et al.; Glucose performs key functions as a signaling molecule in the yeast Saccharomyces cerevisiae . Glucose depletion is known to regulate gene expression via pathways that lead to derepression of genes at the transcriptional level . In this study, we have investigated the effect of glucose depletion on protein synthesis . We discovered that glucose withdrawal from the growth medium led to a rapid inhibition of protein synthesis and that this effect was readily reversed upon readdition of glucose . Neither the inhibition nor the reactivation of translation required new transcription . This inhibition also did not require activation of the amino acid starvation pathway or inactivation of the TOR kinase pathway . However, mutants in the glucose repression (reg1, glc7, hxk2, and ssn6), hexose transporter induction (snf3 rgt2), and cAMP-dependent protein kinase (tpk1(w) and tpk2(w)) pathways were resistant to the inhibitory effects of glucose withdrawal on translation . These findings highlight the intimate connection between the nutrient status of the cell and its translational capacity . They also help to define a new area of posttranscriptional regulation in yeast. Am J Physiol Lung Cell Mol Physiol, 2000 Mar, 278(3), L545 - 51 IGFBP-3 mediates TGF-beta1-induced cell growth in human airway smooth muscle cells; Cohen P et al.; Both insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor-beta (TGF-beta) have been separately shown to have cell-specific growth-inhibiting or growth-potentiating effects . TGF-beta stimulates IGFBP-3 mRNA and peptide expression in several cell types, and TGF-beta-induced growth inhibition and apoptosis have been shown to be mediated through the induction of IGFBP-3 . However, a link between the growth stimulatory effects of TGF-beta and IGFBP-3-induction has not been shown . In this study, we investigated the role of IGFBP-3 in mediating TGF-beta1-induced cell growth using human airway smooth muscle (ASM) cells as our model . TGF-beta1 (1 ng/ml) treatment induced a 10- to 20-fold increase in the levels of expression of IGFBP-3 mRNA and protein . Addition of either IGFBP-3 or TGF-beta1 to the growth medium resulted in an approximately twofold increase in cell proliferation . Coincubation of ASM cells with IGFBP-3 antisense (but not sense) oligomers as well as with an IGFBP-3 neutralizing antibody (but not with control IgG) blocked the growth induced by TGF-beta1 (P < 0.001) . Several IGFBP-3-associated proteins were observed in ASM cell lysates, which may have a role in the cellular responses to IGFBP-3 . These findings demonstrate that IGFBP-3 is capable of mediating the growth stimulatory effect of TGF-beta in ASM cells. Exp Gerontol, 2000 Feb, 35(1), 63 - 70 The influence of oxygen toxicity on yeast mother cell-specific aging; Nestelbacher R et al.; The effect of deleting both catalase genes and of increased oxygen as well as paraquat (a pro-oxidant) on the replicative life span of yeast mother cells has been investigated to test the so-called oxygen theory of aging . This is well established in higher organisms, but has not been extensively tested in the unicellular yeast model system . Life span determinations were performed in ambient air or in a controlled atmosphere (55% oxygen) and an isogenic series of strains deleted for one or both yeast catalases was used and compared with wild type . In the absence of cellular catalase, increased oxygen caused a marked decrease in life span that could be completely reversed by adding 1 mM GSH, a physiological antioxidant, to the yeast growth medium . In a second unrelated strain, the effects were similar although even the wild type showed a decrease in life span when oxygen was increased . The effect could again be compensated by addition of extracellular GSH . Our results show that manipulating the detoxification of reactive oxygen species has a profound effect on yeast aging . These findings are discussed in the light of recent results relating to oxygen toxicity in the aging process of higher organisms. Vet Microbiol, 2000 Feb, 71(3-4), 269 - 79 A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas; Young TF et al.; An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas . Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes . Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface . Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study . The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy . Adherence of M . hyopneumoniae and M . flocculare and damage to the cilia were demonstrated using scanning electron microscopy . The pathogenic M . hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage . The non-pathogenic M . hyopneumoniae strain J did not adhere to mature cilia . Both M . flocculare strains Ms42 and 7160T adhered to mature and budding cilia . No obvious ciliary damage was observed with strain Ms42 . Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T . This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia. Nat Biotechnol, 2000 Mar, 18(3), 304 - 8 Myoseverin, a microtubule-binding molecule with novel cellular effects; Rosania GR et al.; A new microtubule-binding molecule, myoseverin, was identified from a library of 2,6,9-trisubstituted purines in a morphological differentiation screen . Myoseverin induces the reversible fission of multinucleated myotubes into mononucleated fragments . Myotube fission promotes DNA synthesis and cell proliferation after removal of the compound and transfer of the cells to fresh growth medium . Transcriptional profiling and biochemical analysis indicate that myoseverin alone does not reverse the biochemical differentiation process . Instead, myoseverin affects the expression of a variety of growth factor, immunomodulatory, extracellular matrix-remodeling, and stress response genes, consistent with the activation of pathways involved in wound healing and tissue regeneration. Appl Environ Microbiol, 2000 Mar, 66(3), 1120 - 5 Medium-chain fatty acids affect citrinin production in the filamentous fungus Monascus ruber; Hajjaj H et al.; During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals . Analysis of the (13)C-pigment molecules from mycelia cultivated with {1-(13)C}-, {2-(13)C}-, or {1, 2-(13)C}acetate by (13)C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction . Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth . Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone . However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter . Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M . ruber when they were added to the medium . Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones. Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 57 - 64 Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans; Frattini CJ et al.; The effects of naturally-occurring organic compounds on ferrous iron oxidation by the bacterium Thiobacillus ferrooxidans were examined with a view to using these compounds to treat or prevent acid mine/rock drainage . The compounds glucose, cellobiose, galacturonic acid, and citric acid were added to the growth medium of five different strains of the bacterium and growth studies were done to determine whether or not strain differences existed with respect to organic compound sensitivity . The effects of these compounds were compared to the effects of sodium dodecyl sulfate (SDS) an anionic detergent . Each of the compounds tested had an inhibitory effect on the strains of the bacterium and sensitivity to these compounds was strain dependent . All strains appeared to be equally susceptible to SDS . Inhibitory concentrations ranged from 70 mM to >280 mM for glucose, 7.5 mM to 150 mM for cellobiose, 20 mM to 230 mM for galacturonic acid, and 50 mM to 130 mM for citric acid . SDS effectively inhibited iron oxidation for all strains at a concentration of 0.3 mM, the lowest concentration tested . Some naturally-occurring organic compounds, therefore, might be candidates for the growth control of T . ferrooxidans. BJU Int, 2000 Mar, 85(4), 504 - 13 Comparison of marker protein expression in benign prostatic hyperplasia in vivo and in vitro; Fry PM et al.; OBJECTIVE: To use multiple immunofluorescence to compare the in vivo and in vitro expression of tissue-specific proteins in BPH . Materials and methods Pure populations of prostate epithelial and stromal cells were produced using standard methods . Serum-free media for epithelial cells were compared . Co-localization of proteins was compared in frozen-tissue sections and cultured cells by simultaneous multiple immunofluorescence, and recorded using a high-resolution charge-coupled device camera . RESULTS: In contrast to the other serum-free media tested, epithelial cells grew without squamous differentiation or vacuolation in prostate epithelial growth medium (PrEGM, Clonetics, BioWhittaker UK Ltd., Berks, UK) . These cells were predominantly of a basal phenotype, with some cells showing a luminal phenotype . Most of the stromal cells had features of myofibroblasts, but smooth muscle cells and fibroblasts also were present . CONCLUSION: PrEGM is a commercially available serum-free medium in which primary cultures of prostate epithelial cells can be propagated reproducibly . This study provides a comprehensive description of tissue-specific protein expression in BPH in vivo and in vitro . The use of simultaneous multiple immunofluorescence to study co-localization has resulted in a more precise definition of phenotype than has previously been possible, thereby establishing the relevance of the in vitro model system BPH. Exp Neurol, 2000 Jan, 161(1), 67 - 84 Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS; Villa A et al.; The ready availability of unlimited quantities of neural stem cells derived from the human brain holds great interest for basic and applied neuroscience, including therapeutic cell replacement and gene transfer following transplantation . We report here the combination of epigenetic and genetic procedures for perpetuating human neural stem cell lines . Thus we tested various culture conditions and genes for those that optimally allow for the continuous, rapid expansion and passaging of human neural stem cells . Among them, v-myc (the p110 gag-myc fusion protein derived from the avian retroviral genome) seems to be the most effective gene; we have also identified a strict requirement for the presence of mitogens (FGF-2 and EGF) in the growth medium, in effect constituting a conditional perpetuality or immortalization . A monoclonal, nestin-positive, human neural stem cell line (HNSC.100) perpetuated in this way divides every 40 h and stops dividing upon mitogen removal, undergoing spontaneous morphological differentiation and upregulating markers of the three fundamental lineages in the CNS (neurons, astrocytes, and oligodendrocytes) . HNSC.100 cells therefore retain basic features of epigenetically expanded human neural stem cells . Clonal analysis confirmed the stability, multipotency, and self-renewability of the cell line . Finally, HNSC.100 can be transfected and transduced using a variety of procedures and genes encoding proteins for marking purposes and of therapeutic interest (e.g., human tyrosine hydroxylase I) . Mol Microbiol, 2000 Feb, 35(3), 542 - 52 Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress; Krieger S et al.; In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms . To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter . This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline . In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant . In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM) . The depletion of TRYR had no effect on susceptibility to melarsen oxide . The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed. Ann N Y Acad Sci, 1999, 890, 421 - 37 Intracellular survival pathways against glutamate receptor agonist excitotoxicity in cultured neurons . Intracellular calcium responses; Marini AM et al.; Cultured rat cerebellar granule cells are resistant to the excitotoxic effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor agonists under three conditions: 1) prior to day seven in vitro when cultured in depolarizing concentrations of potassium {25 mM}; 2) at any time in vitro when cultured in non-depolarizing concentrations of potassium 5 mM{; and 3) when neurons, cultured in depolarizing concentrations of potassium 25 mM{ for eight days in vitro, are pretreated with a subtoxic concentration of NMDA . The focus of this paper is to determine: a) whether the resistance to excitotoxicity by NMDA and non-NMDA receptor agonists is due to a decreased intracellular calcium Ca++{i response to glutamate receptor agonists in cultured rat cerebellar granule cells; or b) whether Ca++{i levels induced by the agonists are similar to those observed under excitotoxic conditions . Granule cells, matured in non-depolarizing growth medium, treated with glutamate resulted in an increase in Ca++{i followed by a plateau that remained above baseline in virtually all neurons that responded to glutamate . The response was rapid in onset (< 10 sec) and the pattern of response heterogeneous in that cells responsive to glutamate increased their Ca++{i to different extents; some cells did not respond to glutamate . Kainate also produced significant elevations in Ca++{i . The Ca++{i response to glutamate in neurons matured in depolarizing (25 mM K+) growth medium for three days was rapid, transient and heterogeneous, which reached a plateau that was elevated above baseline levels; removing the glutamate markedly reduced the Ca++{i concentration . Activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors by kainic acid produced similar changes in Ca++{i responses . At a time when cultured cerebellar granule cells become susceptible to the excitotoxic effects of glutamate acting at NMDA receptors (day in vitro (DIV) 8) in depolarizing growth medium, glutamate elicited Ca++{i responses similar to those observed at a culture time when the neurons are not susceptible to the excitotoxic effects of glutamate (DIV 3) . Pretreatment of the cultured neurons with a subtoxic concentration of NMDA, which protects all neurons against the excitotoxic effects of glutamate, did not alter the maximal Ca++{i elicited by an excitotoxic concentration of glutamate. Chem Senses, 2000 Feb, 25(1), 93 - 101 Olfactory receptor neurons in partially purified epithelial cell cultures: comparison of techniques for partial purification and identification of insulin as an important survival factor; McEntire JK et al.; Olfactory neurons have the rare property of being replaced throughout life . Factors regulating different developmental stages of olfactory receptor neurons (ORNs) are of great interest, because such factors might be used to extend regeneration in the post-developmental brain and spinal cord . Also, these factors may potentially be exploited to treat various smell disorders arising from changes in the olfactory epithelium . Characterization of trophic factors for ORNs requires cell culture systems that are simple and easy to manipulate . We have compared four different cell culture preparations, using two different enzymes and two different media to develop a simple culture system of olfactory epithelial cells . Our preferred preparation, which produces partially purified olfactory epithelial cultures, uses trypsin dissociation and a serum-free keratinocyte growth medium (KGM) supplemented with insulin . These conditions support ORN survival up to 1 week . They also supported other elements of the olfactory epithelium such as Bowman's gland cells and horizontal basal cells . Olfactory epithelial cells predominate, while contaminating mesenchymal cells (glia and fibroblasts) are present in low numbers . Using these cultures, it was determined that insulin was required for ORN survival in vitro . The simplicity of the epithelial cultures will be useful for further studies of insulin and other ORN trophic factors. Folia Microbiol (Praha), 1999, 44(3), 283 - 8 Effect of killer toxin K1 on yeast membrane potential reported by the diS-C3(3) probe reflects strain- and physiological state-dependent variations; Eminger M et al.; The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S . cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions . Killer toxin K1 brings about changes in membrane potential . In all types of cells tested, viz . in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells . This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration . The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin . In susceptible cells, i.e . in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum lambda max indicating damage to membrane integrity and cell permeabilization . A similar fast red shift in lambda max signifying lethal cell damage was found in heat-killed or nystatin-treated cells. Mol Cell Biol Res Commun, 1999 Sep-Dec, 2(3), 172 - 7 CAMP is involved in transcriptional regulation of delta9-desaturase during Histoplasma capsulatum morphogenesis; Storlazzi A et al.; We have characterized the promoter region of the delta9-desaturase gene from two different strains of the dimorphic fungus Histoplasma capsulatum . Desaturase transcription is regulated in the two phases of growth: it is transcribed in the yeast phase at 37 degrees C, while it is inactive in the mycelial phase at 25 degrees C . Phase transition can be induced by shifting the temperature from 25 to 37 degrees C or by adding cAMP to the growth medium . We have identified a stress-responsive cis element (STRE) responsive to cyclic AMP (cAMP)-signaling pathway and demonstrated that this element acts in H . capsulatum . We have also identified an element, hereafter called DRE (Desaturase Regulatory Element), present in the promoters of the H . capsulatum and S . cerevisiae delta9-desaturase gene . We show that this element is necessary but not sufficient to regulate transcription of the H . capsulatum delta9-desaturase gene. J Biotechnol, 2000 Jan 21, 76(2-3), 253 - 8 An alpha-L-arabinofuranosidase from Penicillium purpurogenum: production, purification and properties; De Ioannes P et al.; Penicillium purpurogenum secretes arabinofuranosidase to the growth medium . Highest levels of enzyme (1.0 U ml(-1)) are obtained when L-arabitol is used as carbon source, while 0.85 and 0.7 U ml(-1) are produced with sugar beet pulp and oat spelts xylan, respectively . By means of a zymogram, three bands with arabinofuranosidase activity have been detected in the supernatant of a culture grown in oat spelts xylan . One of the enzymes was purified to homogeneity from this supernatant using gel filtration (BioGel P-100), cation exchange chromatography (CM-Sephadex C-50), hydrophobic interaction chromatography (phenyl agarose) and a second BioGel P-100 column . The enzyme is a monomer of 58 kDa with a pI of 6.5 . Optimum pH is 4.0 and optimal temperature 50 degrees C . The arabinofuranosidase is highly specific for alpha-L-arabinofuranosides and liberates arabinose from arabinoxylan . The enzyme shows hyperbolic kinetics towards p-nitrophenyl-alpha-L-arabinofuranoside with a K(M) of 1.23 mM . A 36-residue N-terminal sequence is over 70% identical to that of fungal arabinofuranosidases belonging to family 54 of the glycosyl hydrolases . Based on the sequence similarity and other biochemical properties it is proposed that the purified enzyme from P . purpurogenum belongs to family 54. J Biotechnol, 2000 Jan 21, 76(2-3), 245 - 51 Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger; Svetina M et al.; The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13 . Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium . Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway . The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium . The secreted EK(L) was easily purified from other proteins found in A . niger culture supernatant, using ion exchange and affinity chromatography . The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture. J Biotechnol, 2000 Jan 21, 76(2-3), 175 - 83 Effects of growth medium selection on plasmid DNA production and initial processing steps; O'Kennedy RD et al.; Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy . A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB) . Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium . Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis . The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium . It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics . Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage . Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium. J Clin Microbiol, 2000 Feb, 38(2), 733 - 6 Comparison of effects of medium composition and atmospheric conditions on detection of Bilophila wadsworthia beta-lactamase by cefinase and cefinase plus methods; Summanen PH; The influence of growth medium and incubation conditions on the detection of Bilophila wadsworthia beta-lactamase was tested with Cefinase and Cefinase Plus disks . The tests involved aerobic and anaerobic incubation with conventional disk and quantitative tube assays . The production of beta-lactamase was correlated with penicillin G, ampicillin, and ampicillin-sulbactam MICs and inhibition zones on penicillin (2-U) disks . The strains were grown on (i) brucella agar (brucella), (ii) brucella agar supplemented with 1% pyruvate (brucella-pyruvate), and (iii) brucella agar supplemented with 1% taurine (brucella-taurine) . With the aerobic disk assay, 100, 100, and 7% of strains were positive after 30 min from growth on brucella-pyruvate, brucella, and brucella-taurine plates, respectively; of strains grown on brucella-taurine, 54% remained negative by the Cefinase assay, and 23% remained negative by the Cefinase Plus assay at 2 h . In quantitative assays, the strains became positive after 30 min from brucella-pyruvate plates and after 1 h from brucella plates . The intensities of the reactions were strongest with brucella-pyruvate plates under anaerobic test conditions . Anaerobic incubation enhanced beta-lactamase detection of growth on brucella-taurine: at 3 h, 85% of strains were positive in comparison to 38% with aerobic incubation . All beta-lactamase-negative strains were susceptible to penicillin G and ampicillin; all beta-lactamase-positive strains were resistant to ampicillin and, with the exception of two strains, penicillin G . In conclusion, beta-lactamase production correlated with susceptibility to penicillin G and ampicillin . Brucella agar supplemented with 1% pyruvate was the most reliable medium for testing B . wadsworthia beta-lactamase, and anaerobic incubation expedited positive results . Brucella agar supplemented with taurine was unsuitable for B . wadsworthia beta-lactamase testing . Cefinase and Cefinase Plus results were in agreement, but Cefinase Plus yielded faster reactions. Graefes Arch Clin Exp Ophthalmol, 1999 Dec, 237(12), 1001 - 6 Physiological features of primary cultures and subcultures of human retinal pigment epithelial cells before and after cryopreservation for cell transplantation; Valtink M et al.; BACKGROUND: One striking disadvantage of in vitro culturing of human retinal pigment epithelial (RPE) cells is the loss of epithelial differentiation and specific cell function during culture . This may be one of the main reasons for the failure of RPE cell transplantation . The aim of this study was to evaluate cell culture conditions ensuring the maintenance of differentiation and function of RPE cells after subcultivation and storage in liquid nitrogen . METHODS: Enzymatically isolated cells were seeded onto coated culture dishes, cultured with a specially formulated improved growth medium until confluence and then cryopreserved in liquid nitrogen for 16-66 months . HLA class I and II typing was performed before cryopreservation and after thawing . Expression of Ca2+ channels in primary, first-passage and cryopreserved RPE cells was studied using the patch-clamp technique . RESULTS: After cryopreservation no loss of any HLA antigen was detectable in 12 of 14 cell strains studied . Patch-clamp experiments demonstrated that high-threshold L-type Ca2+ channels, which are typical for freshly isolated cells, could be detected in first-passage and cryopreserved RPE cells only when improved culture conditions were employed, not in conventionally cultured cells . The characteristics of these channels showed little change in subcultured cells compared to primary cultures . CONCLUSION: This is the first study showing the maintenance of adult human RPE-specific cell differentiation and characteristics in vitro after primary culture and after cryopreservation using improved cell culture methods . The optimization and quality control of cell culture is an important prerequisite for successful cell transplantation. Pflugers Arch, 2000, 439(3 Suppl), R97 - 9 Isolation, partial length sequence and expression of steroid inducible hps 70 gene from Rhizopus nigricans; Cernila B et al.; Previous studies have shown that filamentous fungus Rhizopus nigricans responds to addition of different steroids into growth medium with induction of hydroxylation system and that some steroids provoke stress response . The purpose of this study was to investigate whether those steroids provoke induction of Hsp70 gene(s), well studied markers of stress response in different cells and organisms . The expression studies of fungal Hsp70 gene(s) using Northern blot analysis showed that fungal hsp70 mRNA was upregulated after treatment of mycelia with deoxycorticosterone and testosterone, but not after exposure to progesterone . In addition, expression of fungal Hsp70 mRNA was elevated after exposure of mycelia to heat shock (32 degrees C), ethanol, heavy metal (CuSO4), and oxidative stressor (H2O2), whereas treatment of mycelia with osmotic stressor (KCl) didn't have any influence on stress protein expression . The partial nucleotide sequence and deduced amino acid sequence homology search revealed that the cDNA clone (lambda hs20/2), isolated from cDNA library prepared from heat shock treated fungal mycelia, contained Hsp70 gene of DnaK subfamily. J Biol Chem, 2000 Feb 4, 275(5), 3637 - 44 Reentry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor . Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase; Suzuki E et al.; Vascular endothelial cells are unique in that they exit from the cell cycle when they come into contact with each other . Although the phenomenon is called "contact inhibition," little is known about the cellular mechanisms involved . Here we show that the phosphatase inhibitor sodium orthovanadate (SOV) induced the reentry of contact-inhibited human umbilical vascular endothelial cells (HUVECs) into the cell cycle and that reentry was associated with activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K)/Akt pathways . SOV stimulated {(3)H}thymidine uptake of contact-inhibited HUVECs in a time- and dose-dependent manner . SOV-induced increase in {(3)H}thymidine uptake was significantly inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by the PI 3-K inhibitor LY294002 . SOV also stimulated the expression of cyclin D1, cyclin E, and cyclin A, and the activity of CDK2 kinase, whereas it decreased the expression of p27(kip1) . In marked contrast, growth media alone did not induce these changes . Furthermore, these SOV-induced changes were abolished by pretreatment with PD98059 and LY294002 . SOV stimulated phosphorylation of ERK and Akt in contact-inhibited HUVECs, while growth media alone did not . This phosphorylation was associated with inhibition of phosphatase activity in the cells . Finally, overexpression of high cell density-enhanced protein tyrosine phosphatase 1 inhibited c-fos and cyclin A promoter activity . Taken together, our results suggest that in contact-inhibited HUVECs, increased phosphatase activity suppressed the ERK and PI 3-K/Akt pathways, resulting in exit from the cell cycle by down-regulation of cyclin D1, cyclin E, and cyclin A and by up-regulation of p27(kip1). Eur J Biochem, 2000 Feb, 267(3), 690 - 702 Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells; Drori S et al.; Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity . We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells . The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied . Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6 . This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR . These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3'-UTR . Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face . Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating approximately 65-90 kDa native reduced folate carrier and a approximately 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation . However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-{3H}MTX and NHS-{3H}folic acid . Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate . This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement . The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 159 - 64 The periodontal pathogen Porphyromonas gingivalis harnesses the chemistry of the mu-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide; Smalley JW et al.; The major haem component in the black pigment of Porphyromonas gingivalis is the mu-oxo bishaem of iron protoporphyrin IX and formation and cell-surface binding of this haem species is proposed as an extracellular buffer against reactive oxidants {Smalley, J.W . et al . (1998) Biochem . J . 331, 681-685} . P . gingivalis cells grown in the presence of the mu-oxo bishaem were protected against H(2)O(2) compared to control cells grown without it . When added to the growth medium, soluble mu-oxo bishaem inactivated H(2)O(2) and supported cell growth . Cells carrying a surface layer of mu-oxo bishaem were less susceptible to peroxidation by H(2)O(2) . Cell-surface haems were slowly destroyed during reaction with H(2)O(2) . Binding of mu-oxo bishaem by P . gingivalis may aid survival during neutrophil attack through inactivation of hydrogen peroxide. Adv Perit Dial, 1998, 14, 149 - 53 New connection method for isolating and disinfecting intraluminal path during peritoneal dialysis solution-exchange procedures; Grabowy RS et al.; Microbiological data have been collected on the performance of a new method of isolating and disinfecting the intraluminal path at the connect/disconnect site of a peritoneal dialysis (PD)-exchange pathway . High-temperature moist-heat (HTMH) disinfection is accomplished by a new device that uses microwave energy to heat the solution contained in the pressure-tight inner lumen of PD connector pairs between the transfer-set connector-clamp and the bag-connector break-away seal . An 85 degrees C (S.D . = 2.4 degrees C, n = 10) rise in solution temperature is seen in 12 seconds, thus yielding temperatures under pressure well over 100 degrees C with starting temperatures of 25 degrees C . Connector pairs were prepared by inoculation of a solution suspension containing at least 10(6) colony-forming units (CFU) of a test micro-organism . Approximately 0.4 mL of solution was contained within the mated connector pair . Using standard D-value determination methods, data were obtained for surviving organisms versus five exposure times and a positive control to obtain a population reduction curve . Four micro-organisms (S . epidermidis, P . aeruginosa, C . albicans, and A . niger) recognized to be among the most prevalent or problematic in causing peritonitis were tested . After microwave heating, the treated solution was aseptically withdrawn from the connector pair using a needle and syringe, plated in growth media, and incubated . Population counts of CFUs after incubation were used to establish survival curves . Results showed a tenfold population reduction in less than 3 seconds for all organisms tested . A 30-second cycle time safely achieves a > 10(8) population-reduction for bacteria and yeast organisms, and a > 10(7) population reduction for fungi . One potential benefit of using this new intraluminal disinfection method is that it may help reduce peritonitis resulting from the even more problematic pathogens such as the gram-negative bacteria and fungal organisms. Can J Ophthalmol, 1999 Dec, 34(7), 379 - 84 Experimental gene therapy for an in vitro model of proliferative vitreoretinopathy; Wong CA et al.; BACKGROUND: Proliferative vitreoretinopathy (PVR) is the leading cause of failure of retinal reattachment surgery . Since a key component of PVR is cell proliferation, we performed a study to examine whether the ribonucleotide-reductase-deficient herpes simplex virus type I (HSV-I) mutant hrR3 can be effective at destroying proliferating retinal pigment epithelial (RPE) cells and thus prevent epiretinal membrane formation and PVR, while sparing nondividing cells, such as neurons . METHODS: Primary cultures of rat RPE cells and rat cortical neurons were infected with 300 microL of hrR3 HSV-I to achieve a multiplicity of infection of 1.0 . After 1 hour at 37 degrees C, 700 microL of growth medium was added to raise the total volume of medium to 1 mL . At 0, 12, 24 and 36 hours the cultures were observed, and the ratio of dead cells to live cells was determined . HSV infection and protein expression were confirmed by a beta-galactosidase histochemical assay or an antihuman HSV-I immunoassay, or both . RESULTS: At 24 hours more than 95% of the RPE cells and neurons stained positively for HSV infection, although beta-galactosidase was expressed predominantly in RPE cells . At 36 hours 72% (standard deviation 2.1%) of the RPE cells were dead . There was no noticeable cell death in the neuronal or mock-infected control cultures . INTERPRETATION: The results suggest that the hrR3 mutant strain of HSV-I can be used to infect and selectively kill actively proliferating rat RPE cells while sparing normal, nonreplicating cells . This model may be used to explore potential therapies for PVR in humans. Hum Gene Ther, 2000 Jan 1, 11(1), 139 - 49 Improved production of adenovirus vectors expressing apoptotic transgenes; Bruder JT et al.; Adenovirus vectors expressing gene products that can induce apoptosis have potential utility in gene therapy applications ranging from the treatment of proliferative diseases to transplantation . However, adenovirus vectors carrying proapoptotic gene products are difficult to produce, as the apoptotic environment is not conducive to adenovirus gene expression and replication . Production of AdFasL/G, an adenovirus vector that expresses high levels of Fas ligand, was severely reduced in the 293 packaging cell line . Increased yields of AdFasL/G were achieved by inclusion of peptide-based caspase inhibitors in the growth medium . However, use of these inhibitors for large-scale production would be difficult and expensive . A screen for gene products that increase the yield of AdFasL/G in 293 cells revealed that the poxvirus serpin CrmA and the adenovirus 14.7K product were able to increase virus yields significantly . Apoptosis induced by AdFasL/G was attenuated in 293CrmA cell lines and virus titers were increased dramatically . However, serial passage of AdFasL/G on 293CrmA cells resulted in the generation of replication-competent adenovirus . To resolve this problem, the CrmA gene was introduced into AE25 cells, an E1-complementing cell line that has limited sequence identity with the vectors . AdFasL/G titers were increased 100-fold on AE25CrmA cells relative to the AE25 cells and RCA contamination was not detectable . In addition, adenovirus vectors that express FADD, caspase 8, and Fas/APO1 were produced efficiently in AE25CrmA and 293CrmA. Indian J Med Res, 1999 May, 109, 182 - 7 Optimizing radiation therapy of brain tumours by combination of 5-bromo-2-deoxy-uridine & 2-deoxy-D-glucose; Kalia VK; The effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60Co-gamma ray induced damage were studied in a human glioma cell line grown as monolayer . Radiation induced micronuclei formation was used as an index of cytogenetic damage . Exponentially growing cells (doubling time 16-20 h) were incubated in the presence of BrdU (0.8 microM, in dark) for 24 h . After removing BrdU, cells were irradiated (1-4 Gy), incubated with or without 2-DG (2-3 h), and grown further (for 18, 24, 30 or 45 h) for assay of damage . It was observed that (i) BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures; (ii) pre-irradiation presence of BrdU increased the gamma-ray induced micronuclei formation; (iii) incubation of irradiated cells under sub-optimal growth conditions {Dulbecco's modified minimal essential medium (DMEM) + 1% serum, or DMEM alone} instead of growth medium (DMEM + 5% serum) progressively decreased micronuclei formation; and (iv) post-irradiation presence of 2-DG (1.25, 2.5, 5 mM, 2-3 h in DMEM + 1% serum) enhanced the radiation damage with and without BrdU treatment at all the time points studied . These observations suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating cells are, at least, partly repairable; (ii) the presence of 2-DG (2DG/glucose > or = 0.25) for short intervals (approximately 2 h), could enhance radiation damage in proliferating brain tumour cells, in the absence as well as presence of BrdU incorporation; and (iii) the combination of 2-DG could reduce BrdU doses required for radiosensitization of brain tumours, reducing, thereby, its toxic side effects. J Vasc Surg, 2000 Jan, 31(1 Pt 1), 181 - 9 Isolation of endothelial cells and their progenitor cells from human peripheral blood; Boyer M et al.; PURPOSE: We have developed techniques to isolate endothelial cell (EC) progenitors from human peripheral and umbilical cord blood . METHODS: Human adult peripheral and umbilical cord blood monocytes were isolated by centrifugation, and progenitor cells were separated with the use of magnetic polystyrene beads that were coated with a monoclonal antibody specific for the CD34 cell-membrane antigen . Cells were propagated in selective media, and developing cultures were immunostained for CD31, CD34, factor VIII, and vascular endothelial growth factor cell receptors . ECs that developed were transfected with a gene for prourokinase and used to line ePTFE grafts, which were evaluated in vitro in a pulsatile flow system . RESULTS: Umbilical cord monocyte cultures demonstrated colonies that resembled ECs at approximately 2 weeks, with growth being best supported by EC growth media plus 20% calf serum with iron . Immunostaining of colonies was positive for CD31 and factor VIII . After 18 days in culture, CD34(+) cells from adult peripheral blood were noted, which had the typical cobblestone appearance of ECs and immunostained positively for CD31 and factor VIII-related antigens . Cultures of umbilical cord-derived cells and adult peripheral blood-derived cells developed complex line formations within 1 week in culture that stained positively for vascular endothelial growth factor receptor-2 . Urokinase-transfected ECs were shown to overexpress urokinase . Prosthetic grafts lined with transfected cells showed 87.33% +/- 4.97% cell adherence after 2 hours in a pulsatile flow system at clinically relevant shear stress . CONCLUSION: We conclude that endothelial progenitor cells can be isolated from human adult peripheral and umbilical cord blood and developed into EC cultures as a source of cells for vascular graft seeding and gene therapy. Nucleic Acids Res, 2000 Feb 1, 28(3), 755 - 61 The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide; Sandman KE et al.; Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display . This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression . Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT) . The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endo-genous tryptophan-inserting opal suppression pathway . Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage . The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec . These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype . We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion. J Bacteriol, 2000 Feb, 182(3), 714 - 22 Succinate:quinol oxidoreductases in the cyanobacterium synechocystis sp . strain PCC 6803: presence and function in metabolism and electron transport; Cooley JW et al.; The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp . strain PCC 6803 . When the organic acid content in the Deltasll1625 and Deltasll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type . A similar analysis for the Deltasll1625 Deltasll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present . Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate . This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking . In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type . Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium. Am J Vet Res, 2000 Jan, 61(1), 86 - 9 Identification of tuberculosis in cattle slaughtered in Mexico; Milian-Suazo F et al.; OBJECTIVES: To determine epidemiologic factors associated with tuberculosis (TB) in dairy cattle slaughtered in 6 important regions for milk production in Mexico . ANIMALS: 2,500 cattle . PROCEDURE: Tissue specimens with lesions typical of TB were obtained during routine inspection of carcasses at abbatoirs between July 1996 and January 1997 . Infection with Mycobacterium organisms was confirmed by histologic examination and bacteriologic culture . Species identification was made by use of selective growth medium, conventional biochemical tests, and radiometric procedures . Epidemiologic information for affected cattle was obtained by personal interviews with cattle dealers and owners . RESULTS: 400 (16%) of 2,500 cattle carcasses had gross lesions typical of TB . Of the 400 infected cattle, 336 (84%) had lesions in > or = 1 lymph node . Infection was confirmed in 87% of cattle with gross lesions by histologic examination, in 77% by bacteriologic culture at a laboratory in the United States, and in 59% by bacteriologic culture at a laboratory in Mexico . Most cattle were adult females in fair to good body condition that came from large herds (> 500 cattle) and were not included in the Mexican TB control program . CONCLUSIONS AND CLINICAL RELEVANCE: Mean prevalence of lesions typical of TB in dairy cattle at 6 locations in Mexico was 16% . Mycobacterium infection was confirmed by various techniques in most lesions . Recognition of typical gross lesions at slaughter may expedite TB control procedures. Radiat Res, 2000 Feb, 153(2), 131 - 43 Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation; DeSimone JN et al.; Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts . However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling . In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest . Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle . However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle . Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited . Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency . Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls . The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures . Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes . The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays. Mol Cell Biol, 2000 Feb, 20(3), 892 - 9 The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source; Huang HL et al.; The proline utilization pathway in Saccharomyces cerevisiae is regulated by the Put3p transcriptional activator in response to the presence of the inducer proline and the quality of the nitrogen source in the growth medium . Put3p is constitutively bound to the promoters of its target genes, PUT1 and PUT2, under all conditions studied but activates transcription to the maximum extent only in the absence of rich nitrogen sources and in the presence of proline (i.e., when proline serves as the sole source of nitrogen) . Changes in target gene expression therefore occur through changes in the activity of the DNA-bound regulator . In this report, we demonstrate by phosphatase treatment of immunoprecipitates of extracts metabolically labeled with (32)P or (35)S that Put3p is a phosphoprotein . Examination of Put3p isolated from cells grown on a variety of nitrogen sources showed that it was differentially phosphorylated as a function of the quality of the nitrogen source: the poorer the nitrogen source, the slower the gel migration of the phosphoforms . The presence of the inducer does not detectably alter the phosphorylation profile . Activator-defective and activator-constitutive Put3p mutants have been analyzed . One activator-defective mutant appears to be phosphorylated in a pattern similar to that of the wild type, thus separating its ability to be phosphorylated from its ability to activate transcription . Three activator-constitutive mutant proteins from cells grown on an ammonia-containing medium have a phosphorylation profile similar to that of the wild-type protein in cells grown on proline . These results demonstrate a correlation between the phosphorylation status of Put3p and its ability to activate its target genes and suggest that there are two signals, proline induction and quality of nitrogen source, impinging on Put3p that act synergistically for maximum expression of the proline utilization pathway. Photochem Photobiol, 1999 Dec, 70(6), 887 - 92 Oxidation of human catalase by singlet oxygen in myeloid leukemia cells; Lledias F et al.; Catalases are oxidized by singlet oxygen giving rise to more acidic conformers detected in zymograms after electrophoresis in polyacrylamide gels . This shift in catalase mobility can be indicative of singlet oxygen production in vivo . Catalase from human cells, as from many organisms, is susceptible to in vitro modification by singlet oxygen . Human myeloid leukemia (U937) cells were treated under different stress conditions and catalase activity and its electrophoretic mobility was monitored . The U937 cells were found to have high levels of catalase activity, as compared to cultured fibroblasts, and to