J Biol Chem, 1990 Apr 25, 265(12), 6606 - 10 Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells; Hara-Kuge S et al.; Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1 . Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes . Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity . Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1 . The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K . LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells . Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells . LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells . Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X . These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS. Cancer Lett, 1990 Apr 20, 50(2), 103 - 7 Residual proliferative capacity in F9 teratocarcinoma stem cell cultures treated with alpha-difluoromethylornithine, an inducer of parietal endoderm differentiation; Wallon M et al.; We have previously shown that inhibition of polyamine biosynthesis by treatment with 5 mM alpha-difluoromethylornithine (DFMO) causes growth arrest and induces differentiation of F9 teratocarcinoma stem cells . The resulting phenotype is similar to that of parietal endoderm, and these differentiated cells possess no apparent proliferative capacity . In the present study, however, it is demonstrated that some of the DFMO-treated cells are not terminally differentiated . Upon a change to DFMO-free growth medium these cells eventually start to proliferate . Using a colony forming efficiency assay, it is estimated that less than 1 out of 200,000 cells retains its proliferative capacity after 6-10 days of DFMO treatment . These cells exhibit no apparent resistance to DFMO, and their population doubling time is similar to that of untreated control F9 cells . Consequently, the possible existence of a small, quiescent, cell population possessing proliferative potential must be taken into account when designing therapeutic protocols for DFMO. Schweiz Rundsch Med Prax, 1990 Apr 10, 79(15), 464 - 7 {Cancer treatment using Dr . Moerman's diet and therapy . Documentation No . 24}; Deplazes G et al.; For prophylaxis of cancer and treatment of manifest cancer Morerman recommends as the basis of his therapy a lactovegetable diet and, in addition, the '8 essential substances': vitamins A, B, C and E, iodine, sulfur, iron and citric acid . At a later stage he also recommends supplementary vitamin D and selenium . The most important aspect is the change in dietary habits required by the diet prescribed by Moerman and the ingestion of the '8 essential substances' in the form of conventional preparations . The daily cost of treatment of a prostatic cancer, for instance, ranges from about Fr . 3.- to Fr . 6.- . Side effects are not mentioned . The diet and therapy were developed by the Dutch physician Dr Moerman (1893-1988) as long ago as the 1930s . The promoters are the iridiologist J . Landman, the nutritional consultant E . Wannee and the writer R . Jochems . All three have written a book on Moerman . In Switzerland, the Lifecare Association endeavours to disseminate this form of therapy . A chronic deficiency of the '8 essential substances' is said to lead to metabolic disturbances, structural and behavioural anomalies of the regeneration tissue and alkalosis, which is claimed to be a fertile soil for the 'symbionts' that can transform healthy cells into cancer cells . Moerman came to this conclusion on the basis of his observations of pigeons . By means of a lactovegetable diet and substitution of the '8 essential substances', this metabolic disorder is said to be reversible, thus robbing the 'symbionts' of their growth medium . The results of the experiments with pigeons have, as far as we know, never been published.(ABSTRACT TRUNCATED AT 250 WORDS) Wei Sheng Wu Xue Bao, 1990 Apr, 30(2), 98 - 104 {Development of a new system for selection of A . foetidus transformed with foreign genes by using thymidine kinase gene as a marker and expression of HBsAg gene in A . foetidus}; Liu HD et al.; A new system for selection of transformed Aspergillus foetidus was reported . In this system, TK- A . foetidus which were constructed by homologous recombination of mutated TK gene of vaccinia virus with TK gene of A . foetidus were screened by adding BUdR in agar plates . Conditions for screen of TK+ A . foetidus strain, transformation of A . foetidus and selection of transformed TK- A . foetidus have been studied . By using this system, several transformed A . foetidus which contained HBsAg gene derived bf a promoter H8 cloned from genomic DNA of A . foetidus were isolated . It was demonstrated that HBsAg gene was integrated into the chromosome DNA of A . foetidus by Southern blot after many passages of spores . ELISA showed that HBsAg was positive in the growth medium (p/n = 20) . The 22 nm particles which were very similar to the HBsAg particles in human serum were found in the growth medium by immunoelectromicroscope . Western blot also gave the specific bands . All these data showed that HBsAg gene was expressed in A . foetidus and the products were secreted into the growth medium . The selection system using TK gene as marker could generally be used to study the expression of foreign gene in A . foetidus. In Vitro Cell Dev Biol, 1990 Apr, 26(4), 379 - 87 Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions; McLane JA et al.; 1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes . The growth and differentiation of keratinocytes in the presence of 1,25-(OH)2-D3 using serum-free media formulations has been described previously . This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)2-D3 . Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts . The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors . Keratinocyte cell growth was greatest in the KGM medium . Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)2-D3 . The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)2-D3 . The antiproliferative effect of 1,25-(OH)2-D3 was not altered by varying the extracellular calcium levels from 0.25 to 3 mM . The antiproliferative activity of 1,25-(OH)2-D3 is attenuated in cells at low density . Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)2-D3 to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer . These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)2-D3, but can be used to assay other agents that modulate keratinocyte proliferation. Cancer Res, 1990 Apr 1, 50(7), 2123 - 7 Suppression of radiation-induced neoplastic transformation of human cell hybrids by long term incubation at low extracellular pH; Mendonca MS et al.; We have previously reported that, when 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer was used in the growth medium to control pH fluctuations during the 21-day expression period of our human cell hybrid (HeLa x skin fibroblast) transformation assay, the yield of radiation-induced neoplastically transformed foci after 7 Gy of gamma-irradiation was suppressed . We now demonstrate that the observed suppression is not related to the presence of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer per se but rather is a function of the growth medium pH . Detailed studies reveal that incubation of the irradiated cells during the entire 21-day expression period at pH 6.7-6.8 versus pH 7.0-7.2 significantly suppressed the transformation frequency after 7 Gy, from 4.4 x 10(-4) to 4.6 x 10(-5) (accumulated data) . The endpoint fraction of flasks containing foci was also significantly reduced at the lower pH . Suppression was evident whether the growth medium pH was lowered from pH 7.0-7.2 to pH 6.7-6.8 by medium exchange on day 0, 1, or 9 or even up to 15 days post-irradiation . Growth curves revealed that the population doubling time of the cells is extended and the unirradiated and irradiated plating efficiencies are lowered by long term low pH exposure . We discuss possible mechanisms for the observed suppression, in terms of the influence of low extracellular pH on cell turnover, repair of radiation damage, cell toxicity, and activity of cellular proteases. J Antibiot (Tokyo), 1990 Apr, 43(4), 411 - 6 The mode of antifungal action of (S)2-amino-4-oxo-5-hydroxypentanoic acid, RI-331; Yamaguchi M et al.; An antifungal amino acid antibiotic, (S)2-amino-4-oxo-5-hydroxypentanoic acid (RI-331) isolated from Streptomyces sp., inhibited the biosynthesis of protein to a greater extent than that of RNA or DNA in growing Saccharomyces cerevisiae cells . Polypeptide biosynthesis in a cell-free system from the yeast was refractory to the antibiotic, suggesting the possibility that the biosynthesis of one or more amino acids might be inhibited . Intracellular amino acid pools, particularly those of methionine, isoleucine and threonine were significantly reduced when yeast cells were incubated in the presence of RI-331 . Consistent with this, the growth-inhibitory activity of RI-331 was markedly reversed by the addition of these amino acids into the growth medium, and an even greater effect was exerted by homoserine which works as a common metabolic precursor for these amino acids in yeasts . It looks likely therefore that the inhibition of biosyntheses of some or all of these amino acids by RI-331 is primarily responsible for overall inhibition of protein biosynthesis in yeasts, ultimately leading to cytostasis . This possible mechanism of RI-331 action appears to explain favorably the selective toxicity of the antibiotic against yeasts, since mammalians lack enzymatic systems for synthesizing methionine, isoleucine and threonine which are required as essential amino acids for growth. Appl Environ Microbiol, 1990 Apr, 56(4), 1004 - 11 Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations; Luli GW et al.; The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities . Of the E . coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested . In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1 . Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1 . The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E . coli JM105, JM105(pOS4201), and JRG1061 . These data indicated that the growth of the E . coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose . A model for the inhibition of growth of E . coli by acetate was derived from these experiments to explain the inhibition of acetate on E . coli strains at neutral pH. J Bacteriol, 1990 Apr, 172(4), 1846 - 52 Characterization of malT mutants that constitutively activate the maltose regulon of Escherichia coli; Dardonville B et al.; The expression of the maltose regulon of Escherichia coli is controlled by a transcriptional activator, the product of the malT gene, and is induced by the presence of maltose or maltodextrins in the growth medium . We isolated eight mutants with mutations in malT which lead to constitutive expression of the regulon . The nucleotide sequences of the mutated genes revealed that the eight mutations are clustered in two small regions in the first one-third of the malT gene . Two mutated MalT proteins (corresponding to a mutation in each cluster) were purified and examined for in vitro activation of the MalT-dependent malPp promoter . Whereas wild-type MalT activity was absolutely dependent upon the presence of maltotriose, even at high protein concentrations, both mutated proteins were partially active in the absence of this sugar . Indeed, while the activity of the mutated proteins was still increased by maltotriose at low protein concentrations, the proteins were fully active in the absence of maltotriose at high protein concentrations . Both proteins exhibited a fivefold-higher affinity for maltotriose than the wild-type protein did. J Rheumatol, 1990 Apr, 17(4), 515 - 20 The effect of synovial fluid and serum on the growth of calcium hydroxyapatite crystals; Campion GV et al.; The presence or absence of natural crystal growth inhibitors in joint tissues and fluids may be important in the pathogenesis of several arthropathies . Synovial fluid (SF) has therefore been examined for inhibitors of seeded hydroxyapatite growth rate (Vo) using a pH stat system . Pretreatment of seed crystals with SF reduced growth (Vo = 56 +/- 5, control 117 +/- 141 mol/base/min/g hydroxyapatite, p less than 0.001) . Addition of small amounts of serum or SF to the growth medium caused a dose dependent growth inhibition (0.04% SF reduced Vo by 16%) . Pretreatment with protease, but not hyaluronidase, abolished this activity and gel filtration localized it to the 55-80 kDa fraction . A macromolecular factor(s) with potent inhibitory activity has therefore been detected. J Bacteriol, 1990 Apr, 172(4), 2079 - 87 Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli; Lee JH et al.; A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase . This phenotype was suppressed by supplementing the media with molybdate . Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport . The molR gene is located at 65.3 min in the E . coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished . The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min) . The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media . The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium . In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient . In low-sulfur medium, this value decreased to about 50 nM . The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity . These data suggest that in the absence of the high-affinity molybdate transport system, E . coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system. Prostaglandins Leukot Essent Fatty Acids, 1990 Apr, 39(4), 283 - 90 Leukotriene C4 is an essential 5-lipoxygenase intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells; van Hilten JA et al.; Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately . LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively . Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM . Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM . The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages . Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity . Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4 . This implies that besides LTC4, increased cytosolic {Ca2+} is required for A23187 induction of macrophage cytostatic activity. Mutat Res, 1990 Apr, 243(4), 273 - 80 Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B; Watanabe K et al.; Mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E . coli B . The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants . This effect was lower in an alkA mutant, but was restored in an alkA umuC double mutant . By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant . It was necessary to add simultaneously MNNG and o-vanillin to the growth medium . Further investigations were conducted on the induction of ada and umuC genes using ada'-lacZ' and umuC'-lacZ' plasmids . o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene . In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin . The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine. Membr Biochem, 1990 Apr-Jun, 9(2), 91 - 106 Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence {corrected}; Khatami M; Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied . Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process . At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics . The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA . At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased . The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger) . The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased . Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake . Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively . Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose . Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI . However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake . These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations . Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s) . Glucose influenced the uptake of MI in a complex manner . The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence . High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS) J Virol, 1990 Apr, 64(4), 1429 - 36 Biological activities of a synthetic peptide composed of two unlinked domains from a retroviral transmembrane protein sequence; Wegemer DE et al.; We report several biological activities of a synthetic peptide whose sequence contains the highly conserved region of feline leukemia virus transmembrane protein (TM) synthetically linked to another short TM-derived sequence particularly rich in polar positive residues . This 29-amino-acid peptide blocked {3H}thymidine uptake 30 to 50% by concanavalin A-stimulated CD4(+)--but not CD8(+)-enriched murine splenocytes . Maximal suppression was detected at 12.5 micrograms (3 microM) to 75 micrograms (19 microM) per ml of growth medium; stimulation of {3H}thymidine uptake was observed at higher peptide concentrations . The synthetic peptide inhibited but did not stimulate {3H}thymidine uptake by mitogen-activated thymocytes and antibody production by splenocytes as determined in a liquid hemolytic plaque assay . Similarities are reported between a consensus sequence of diverse retroviral TMs and a region of alpha interferons shown by others to be important for antiviral and cytostatic properties . The TM sequence-derived synthetic peptide blocked in a nontoxic and sequence-specific manner the release of murine leukemia virus from two chronically infected cell lines . We suggest that some of the biological effects of retroviral TM are mediated through a common pathway shared with alpha interferons. Appl Microbiol Biotechnol, 1990 Apr, 33(1), 66 - 71 Phenol-induced membrane changes in free and immobilized Escherichia coli; Keweloh H et al.; Membranes of Escherichia coli cells grown in the presence of phenol were examined after isolation of the cytoplasmic and outer membrane fractions . Both membrane types showed reduced lipid-to-protein ratios compared to cells grown without phenol . Phenol-induced differences in the expression of individual proteins of the inner membrane were established . Different proteins of the outer membrane, probably involved in the uptake of iron, were expressed in smaller quantities after phenol addition . Growth in the presence of phenol increased the respiratory activity of the cytoplasmic membrane, whereas the direct inhibition of O2 consumption by phenol was not affected by the presence of this compound in the growth medium . E . coli cells grown entrapped in calcium alginate showed low lipid-to-protein ratios even without phenol in the growth medium . Immobilization of cells also markedly changed the protein pattern of the outer membrane. J Biol Chem, 1990 Mar 25, 265(9), 5219 - 25 Fibronectin levels are enhanced in human fibroblasts overexpressing the c-sis protooncogene; Allen-Hoffmann BL et al.; We studied human dermal fibroblasts transfected with a human c-sis cDNA (coding for the platelet-derived growth factor B-chain) . Dermal fibroblasts overexpressing c-sis exhibited a stellate morphology with focus formation, enhanced colony formation in methylcellulose-containing growth medium, and increased levels of soluble and extracellular matrix-associated fibronectin . Gene expression of fibronectin was enhanced 10-fold in c-sis-overexpressing fibroblasts relative to controls . Pro-alpha 1 (I) collagen mRNA was not increased in these same c-sis-overexpressing fibroblasts . Transforming growth factor beta 1 treatment of c-sis-transfected cells caused a modest increase (77%) in fibronectin mRNA levels with no increase in soluble fibronectin production after 24 h . In contrast, transforming growth factor beta 1 caused at least a 10-fold increase in fibronectin mRNA and a 2-fold increase in soluble fibronectin from medium conditioned by control fibroblasts . Transforming growth factor beta 1 increased pro-alpha 1 (I) collagen mRNA approximately 3-fold in both control and c-sis-transfected fibroblasts . These studies reveal that a primary biological function of the platelet-derived growth factor B-chain is upregulation of fibronectin gene expression and extracellular matrix formation . The anchorage-independent phenotype of c-sis-overexpressing cells was blocked by the cell adhesion sequence of fibronectin, Arg-Gly-Asp-Ser . Our results demonstrate that interaction of cells with extracellular adhesion receptors is necessary for proliferation in semisolid medium even when cells are overproducing growth factors known to act via autocrine stimulation. FEMS Microbiol Lett, 1990 Mar 15, 56(3), 289 - 93 Osmoregulatory expression of porin genes in Escherichia coli: a comparative study on strains B and K-12; Mizuno T et al.; Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium . In contrast, E . coli B produces only the OmpF porin, regardless of the medium osmolarity . In this study, it was revealed that there is an extensive deletion within the ompC locus of the E . coli B chromosome . Cloning and nucleotide sequencing of the regulatory gene, ompR, of E . coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E . coli K-12 . It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E . coli B. Mech Ageing Dev, 1990 Mar 15, 52(2-3), 287 - 94 Very low ATP/ADP ratios with aging of the natural death senescence mutant of Neurospora crassa; Pall ML; The natural death (nd) mutant of the fungus Neurospora crassa, unlike the wild-type, undergoes an aging process, which leads to the cessation of growth . It is shown here that the ATP/ADP ratio of the mutant declines with age to about 3:1 whereas other strains of Neurospora in the same growth medium maintain ratios of about 8 to 9:1 . The decline in ATP/ADP ratio is not caused by the cessation of growth of the mutant . The results suggest, rather, that the cessation of growth may be caused, in part or in whole, by the defect in energy metabolism that produces the low ATP/ADP ratio . They support the hypothesis that defects in mitochondrial energy metabolism may be an important contributing factor to the aging process. Mikrobiologiia, 1990 Mar-Apr, 59(2), 197 - 204 {Changes in the energy indices of Escherichia coli during exhaustion and renewal of glucose and ammonia supply as a factor responsible for the coupling of energy and constructive types of metabolism}; Tkachenko AG; The shift down of glucose in the growth medium lowered the energetic status of cells whereas that of ammonium elevated it, which was indicative of their specific effect on metabolism . The shift up of glucose within the first four seconds promptly increased the intracellular ATP pool, the energy charge and the ATP/ADP ratio up to values characteristic of growth, while the addition of ammonium after its exhaustion resulted in the opposite effect . The described changes are typical of an incomplete coupling between energetic and constructive metabolic types in E . coli. Lab Invest, 1990 Mar, 62(3), 314 - 24 Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model; Valyi-Nagy IT et al.; The morphologic and antigenic phenotype of normal human melanocytes and keratinocytes was investigated in monolayer and 3-dimensional cultures in an effort to develop an epidermal model that resembles the normal human epidermis . When cultured for several passages in optimal growth medium, pure cultures of either cell type could be established as demonstrated by light and electron microscopy and with monoclonal antibodies defining melanocyte- and keratinocyte-associated antigens . Three-dimensional growth of keratinocytes on polycarbonate filters was induced by increasing calcium concentrations in the culture medium and exposing cultures to air . After 30 to 35 days incubation, the 3-dimensional keratinocyte cultures reached a total of 12 to 25 layers and keratinocytes of various stages of differentiation formed three morphologically and antigenically different strata . The basal layer of these constructs consisted of ovoid cells with desmosomes and hemidesmosome-like structures . These cells expressed low molecular weight cytokeratins similar to basal cells in situ . The intermediate layer, representing the stratum spinosum in situ, contained flat cells with keratohyaline granules and many desmosomes . These cells expressed gp 80 kilodaltons, gp 40 to 50 kilodaltons, involucrin, and filaggrin . The upper layer, the stratum corneum equivalent, contained large, flattened cells with keratohyaline granules . The majority of these cells were anucleate . When melanocytes were cocultured with keratinocytes in monolayer or in epidermal reconstructs, they assumed a multidendritic morphology and donated pigment to surrounding keratinocytes . The majority of pigmented cells localized singly within the basal layer of the reconstructs and their dendrites were intimately associated with keratinocyte plasma membranes . Pigment donation to keratinocytes appeared to occur through the uptake of melanosome-containing dendrite fragments and phagocytosis of individual melanosomes by keratinocytes . It is hypothesized that keratinocytes produce unique microenvironmental factors that regulate the melanocytic phenotype. Exp Cell Res, 1990 Mar, 187(1), 143 - 9 Complete transformation of embryonal rat fibroblasts by polyomavirus occurs during passage in vitro; Martens I et al.; The tumorigenicity of secondary rat embryo fibroblasts transfected with a plasmid harboring a replication origin-defective polyomavirus was found to increase during in vitro propagation . Thus, polyomavirus-transfected cells were found to be more than 10,000-fold more tumorigenic when injected into syngenic rats at 3 months after transfection compared to those injected at an earlier time point . Furthermore, most clones of polyomavirus-transfected cells did not grow in semisolid medium at 52 days after transfection but did grow at 95 days . Addition of glucocorticoid hormones, but not of 25% fetal calf serum, to the growth medium of the early passage cells resulted in limited anchorage-independent growth . An altered level of expression of a number of proteins was found in cells analyzed at different times after transfection . Notably, the expression of a component of the actin filament system, tropomyosin 2, was shown to decrease during growth in vitro . The development of a more fully transformed phenotype at late passages correlated with loss of the requirement for large T-antigen for growth . Thus, cells transfected with a polyomavirus mutant encoding a thermolabile large T-antigen did not grow at the restrictive temperature at 6 weeks after transfection, but grew well at 5 months after transfection . We suggest that these phenomena may be explained by assuming that establishment of rodent fibroblasts, and thereby sensitivity to transformation by middle T-antigen, is not an immediate consequence of expression of large T-antigen but occurs after a period of growth in vitro. Mikrobiologiia, 1990 Mar-Apr, 59(2), 283 - 8 {Heterogeneity of Escherichia coli population during induced autolysis}; Akaizin ES et al.; Escherichia coli M-17 autolysis was induced by eliminating nutrition sources from the growth medium and exerting a shock with EDTA . The overall cell number, the optical density of the cell suspension, the number of colony-forming units (CFU), and {3H}uracil incorporation into the cells were analysed in the course of autolysis . The number of CFU was found to drop down faster than the overall cell number in the process of autolysis . The population of E . coli was shown to be heterogeneous in its sensitivity to the induction of autolysis, and some nonlysed cells were still metabolically active . When the rate of autolysis was highest in some cells of the population, the labeled precursor was found to be incorporated into the TCA-soluble and TCA-insoluble fractions of nonlysed cells . The overall cell number, the optical density of the cell suspension, and the number of CFU increased 96 h after the induction of autolysis . The authors discuss what is the role played by the heterogeneity of an E . coli population in its adaptation to EDTA-induced autolysis. Arch Biochem Biophys, 1990 Mar, 277(2), 263 - 7 Membrane lipid composition, fluidity, and surface charge changes in response to growth of the fresh water cyanobacterium Synechococcus 6311 under high salinity; Khomutov G et al.; The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 6311 on components of the cytoplasmic membranes and thylakoids was investigated . Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium . Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes . The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment. J Bacteriol, 1990 Mar, 172(3), 1464 - 9 Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus; Sparling R et al.; Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2 . Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis . The ability to metabolize formate at high rates was observed only in cells previously grown on formate . Two such strains were distinguished: strain F and strain HF . Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2 . Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate . Initial adaptation of strain H to growth on formate was preceded by a long lag . The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings . Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium . High levels of formate dehydrogenase were also constitutive in strain F . Only low formate dehydrogenase activities were observed in strain H . High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2 . In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase. Biophys J, 1990 Mar, 57(3), 621 - 6 Deviation from homeoviscous adaptation in Escherichia coli membranes; Parola AH et al.; The process by which an organism changes the composition of its membranal fatty acids in response to growth temperature, so as to maintain optimal membrane functioning, is known as homeoviscous adaptation (HA) . One expression of HA is the constancy of the fluorescence polarization (P) of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of cells grown at various temperatures . The P of DPH in the membranes of Escherichia coli was shown by us to be inversely proportional to bacterial growth rate on different carbon sources . This result, implying failure of HA, is now complemented by measurements of DPH lifetimes, which indicate that the dominant variables contributing to the drop in P are (a) the order parameter of the membrane, which goes down, and (b) the fluidity, which may slightly increase . These are then the changes induced by enhanced growth rate . Two additional effects, cell membrane permeability and sensitivity to thermal shock, determined by the diffusion of o-nitrophenylgalactoside (ONPG) and by exposure to 52 degrees C, respectively, are reported to increase with growth rate . We can now conclude that there is a deviation from the principle of HA in E . coli grown at various rates, brought about by controlling the growth media at constant temperatures. Biomed Sci, 1990 Mar, 1(3), 295 - 9 Mobilisation of arachidonic acid in human A431 cells in the presence of epidermal growth factor, calcium ions, and cyclic AMP; Kudryavtseva NG et al.; Epidermal growth factor (EGF) at a concentration of 5 x 10(-11)-10(-8) M invoked mobilisation of arachidonate in cultured A431 human epidermal carcinoma cells and release of a certain proportion of free arachidonic acid and its derivatives into the growth medium . At a concentration of 2 x 10(-8)-4 x 10(-5) M, Ca2+ ions enhanced mobilisation of arachidonate and induced virtually total release of arachidonic acid and its derivatives into the medium . EGF at a concentration of 10(-8) M and Ca2+ ions at a concentration of 2 mM in the presence of calcium ionophore A23187 were found to have an additive effect on the release of arachidonic acid . Release of arachidonic acid by Ca2+ ions was inhibited by 30%-50% in the presence of 10(-4) M dibutyryl-cAMP (Bt2cAMP), isoproterenol, or noradrenaline . There was a 35%-55% increase in the release of arachidonate elicited by 10(-8) M EGF in the presence of 10(-4) M Bt2cAMP, noradrenaline, or isoproterenol . It is concluded that A431 cells contain two independent pathways for the mobilisation of arachidonic acid: a Ca(2+)-dependent pathway and an EGF-dependent pathway. Int J Cancer, 1990 Feb 15, 45(2), 372 - 5 Characterization of the synergistic effect of insulin and transferrin and the regulation of their receptors on a human colon carcinoma cell line; Watkins LF et al.; The human colon carcinoma cell line, HCT 116, can be grown in chemically defined media in the absence of exogenous growth factors . The addition of transferrin and insulin will significantly stimulate growth . The interaction of these growth factors with their receptors was studied to determine whether the synergistic action of insulin and transferrin on growth involved alterations in the growth-factor receptors . Redistribution of the transferrin receptor occurred in the presence of transferrin or transferrin plus insulin . The presence of insulin in the growth media resulted in occupation of cell-surface insulin receptors without a reduction in total insulin binding . Addition of transferrin with insulin resulted in a decrease in insulin binding to its receptor, with no alteration in receptor affinity . It appears that transferrin plays a role in regulating the insulin receptor and that this may contribute to the synergistic effect of insulin and transferrin on growth. Mol Cell Biol, 1990 Feb, 10(2), 643 - 52 HOL1 mutations confer novel ion transport in Saccharomyces cerevisiae; Gaber RF et al.; Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have a functional histidinol dehydrogenase . Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the ability of cells to take up histidinol from the medium . Second-site mutations linked to HOL1-1 further increase histidinol uptake . HOL1 double mutants and, to a lesser extent, HOL1-1 single mutants show hypersensitivity to specific cations added to the growth medium, including Na+, Li+, Cs+, Be2+, guanidinium ion, and histidinol, but not K+, Rb+, Ca2+, or Mg2+ . The Na(+)-hypersensitive phenotype is correlated with increased uptake and accumulation of this ion . The HOL1-1-101 gene was cloned and used to generate a viable haploid strain containing a hol1 deletion mutation (hol1 delta) . The uptake of cations, the dominance of the mutant alleles, and the relative inability of hol1 delta cells to take up histidinol or Na+ suggest that hol1 encodes an ion transporter . The novel pattern of ion transport conferred by HOL1-1 and HOL1-1-101 mutants may be explained by reduced selectivity for the permeant ions. Endocrinology, 1990 Feb, 126(2), 1173 - 82 Casein accumulation in mouse mammary epithelial cells after growth stimulated by different hormonal and nonhormonal agents; Levay-Young BK et al.; Mammary epithelial cells obtained from virgin mice were cultured in collagen gel with linoleic acid-containing serum-free growth medium supplemented with hormonal (PRL and progesterone, epidermal growth factor, somatomedin-C) or nonhormonal (lithium ion, phosphatidic acid containing phospholipid liposomes) growth stimulating agents . The phenotypes of the resulting progeny cells were compared by examining the ultrastructure, immunohistochemical staining for luminal epithelial and myoepithelial cells and casein, and assessing the quantity of biochemically detectable alpha- and beta-casein . Although there are some differences in ultrastructure and immunostaining in the progeny cell populations induced by different growth-promoting agents, all the cultures were able to accumulate alpha- and beta-casein on subsequent stimulation by PRL and linoleic acid in the second phase of culture . Since, in vivo, luminal epithelial cells of the mammary gland are the only cell type capable of synthesizing milk products, these results indicate that all the different growth stimulants, hormonal and nonhormonal, result in the predominant proliferation of luminal-type epithelial cells . These results have important implications for studies of the mechanism of growth control in and transformation of mammary epithelial cells. Pharmacol Toxicol, 1990 Feb, 66(2), 115 - 20 Acute inhibition of human renal tubular cell growth by cyclosporin A; Blaehr H et al.; Human proximal tubular cells were isolated and grown in culture for three passages . The proliferation of these cells were inhibited by the immunosuppressive drug cyclosporin A in dose dependent manner in the range of 250 to 2000 ng/ml growth medium . The cultures with low cell density were more sensitive to cyclosporin A compared to the cultures with high density, measured by the incorporation of 3H-thymidine . The toxic effect of cyclosporin A on cells isolated from patients treated with cyclosporin A, did not differ from cells isolated from normal tissue . The calcium channel blocker, verapamil, reversed the inhibitory effect of low concentrations of cyclosporin A on cell proliferation . The electron microscopy showed that cells treated with cyclosporin A, had severe morphological alterations with rounded mitochondria and giant vesicles in the cytoplasma . The results support the hypothesis that the toxic effect of cyclosporin A may be mediated through an increased Ca++ influx into the proximal tubular cells. Int J Neurosci, 1990 Feb, 50(3-4), 131 - 5 Trophic effects of enkephalin, beta-endorphine and dynorphine on ventral spinal cord in culture; Iwasaki Y et al.; We studied trophic effects of enkephalin (ENK), beta-endorphine (END) and dynorphine (DYN) on explanted cultures of ventral spinal cord from 13-14 day old rat embryo . The addition of each of these three neuropeptides to the growth medium caused no changes in neurite extension and in increased number of glial cells compared to control samples . The results indicate that neurite appearance is neither prompted nor inhibited by addition of ENK, END and DYN . It is considered that ENK, END and DYN are not growth factor of cultured ventral spinal cord of rat embryo. Cell Growth Differ, 1990 Feb, 1(2), 63 - 71 Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor; Wellstein A et al.; We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13) . These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth . The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth . To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene . In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice . A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin . We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo. J Interferon Res, 1990 Feb, 10(1), 13 - 23 Effects of the copper chelators diethyldithiocarbamate and bathocuproine sulfonate on interferon and its antiviral state; Calvert JG et al.; Treatment of mouse L cells with two structurally unrelated chelators of copper ions, diethyldithiocarbamate (DDC) or bathocuproine sulfonate (BCS), completely inhibited the ability of interferon (IFN) to inhibit mengovirus growth . However, mengovirus virions were inactivated by incubation with DDC, and DDC induced a generalized inhibition of cellular RNA and protein synthesis, possibly combined with the inactivation of one or more specific enzymatic activities involved in the establishment or maintenance of the antiviral state . In contrast, BCS had no effect on either cell or virus growth . BCS combined with trace copper ions in the growth medium and the resulting complex prevented IFN from interacting properly with cellular receptors, apparently by binding noncovalently to the IFN molecules . In some cases, IFN was irreversibly inactivated, probably due to oxidation of essential cystein, tyrosine, or tryptophan residues by the (BCS)2Cu2+ complex . BCS was at least as effective as anti-IFN antibody at neutralizing cell-bound IFN, and has a number of advantages over it. J Cell Physiol, 1990 Feb, 142(2), 272 - 83 Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells; Tuder RM et al.; The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP . Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line . This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels . Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML) . Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells . Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells . In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells . These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells . Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature. Trends Biotechnol, 1990 Feb, 8(2), 46 - 52 Bioconversions of aliphatic compounds by Pseudomonas oleovorans in multiphase bioreactors: background and economic potential; Witholt B et al.; Pseudomonas oleovorans can grow on linear alkanes and alkenes in the hexane to dodecane range by virtue of enzymes encoded by the alk genes . By introducing selected alk genes into Pseudomonas strains and by supplying alkanes in the growth medium as a bulk liquid phase, specific alkane oxidation products can be accumulated in the alkane phase . We review the genetics and enzymology of the alk system and the potential of bioconversions in two-liquid-phase bioreactors, and suggest that such systems might eventually allow the biotechnological production of intermediate value compounds. Genetics, 1990 Jan, 124(1), 39 - 55 Consequences of growth media, gene copy number, and regulatory mutations on the expression of the PRB1 gene of Saccharomyces cerevisiae; Moehle CM et al.; Glucose represses PRB1 expression at the level of transcription . However, release from glucose repression initially does not result in accumulation of protease B (PrB) activity despite transcriptional derepression . PrB activity accumulates only upon a second transcriptional derepression as the cells approach stationary phase . Increasing the PRB1 gene dosage on 2 mu-based plasmids does not overcome glucose repression . Glucose-mediated repression of PRB1 is not subject to the same genetic controls as SUC2 . Mutation of the HXK2 gene, which confers glucose-insensitive expression of secreted invertase, had no effect on PRB1 expression at the level of PrB activity . Strains bearing a mutation in any of the SNF1-SNF6 genes cannot derepress secreted invertase synthesis, but did derepress PrB synthesis when grown in the absence of glucose . Mutation of the SNF2 or SNF5 gene led to accumulation of PrB activity to levels ten times that of wild type . Polymorphism for a suppressor gene was observed: in snf5-bearing strains, one allele of this suppressor gene resulted in elevated levels of PrB and the other allele resulted in wild-type levels of PrB; neither allele suppressed the Suc- phenotype of the snf5 mutant . Re-examination of published data on SUC2 expression in snf2 and snf5 mutants and examination of PRB1 expression in these mutants paradoxically suggest that the SNF2 and SNF5 gene products might act as negative regulators of gene expression. Antonie Van Leeuwenhoek, 1990 Jan, 57(1), 37 - 41 Dimorphism of Benjaminiella poitrasii: isolation and biochemical studies of morphological mutants; Khale A et al.; The yeast-mycelium dimorphism of the genus Benjaminiella poitrasii has been investigated . To understand the mechanism of dimorphism two stable yeast-phase mutants (Y-1 & Y-2) and one slow growing mycelial mutant (M-1) of B . poitrasii were isolated after NTG treatment of parent strain spores and studied for their biochemical characteristics . Effects of (i) kind and concentration of carbon source, (ii) presence of complex organic nitrogen and (iii) C:N ratio in the growth medium on the morphology of parent and mutant strains were carried out at 28 degrees C under shaking conditions . Ethanol induced morphological change and its reversal were studied in all the strains in order to elucidate the possible mechanism of morphogenesis. Can J Microbiol, 1990 Jan, 36(1), 15 - 23 Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil; Yarden O et al.; The relationship between chemical structure and the enhancement of microbial degradation of three benzimidazole compounds in soil was determined . Preapplication of methyl benzimidazole-2-ylcarbamate (carbendazim or MBC), 2-aminobenzimidazole (2AB), and benzimidazole enhanced their degradation upon repeated application (self-enhanced degradation) . MBC and 2AB cross-enhanced the degradation of each of these two compounds, whereas benzimidazole did not enhance the degradation of MBC . Thiabendazole (TBZ) did not enhance its own degradation or cross-enhance the degradation of MBC . No increase in the number of MBC-degrading fungi or in the capacity of soilborne fungi to degrade MBC was detected in soil exhibiting enhanced MBC degradation (MBC-history) . A sharp increase in esterolytic activity in the microsomal fraction of Alternaria alternata capable of degrading MBC in culture was induced by the presence of MBC in the growth medium . 2AB was the main metabolite of MBC that accumulated in A . alternata cultures and in cell-free preparations . MBC was degraded much faster by mixed bacterial cultures that originated from MBC-history soil than in cultures from MBC-nonhistory soil . Fluctuations in the MBC degrading capacity of mixed bacterial cultures occurred during repeated subculturing of the mixed culture . Inoculation of nonhistory soil with mixed bacterial cultures resulted in enhanced MBC degradation, whereas inoculation with A . alternata did not enhance MBC degradation . It is suggested that while fungi contribute to MBC dissipation in soil, bacteria have a greater role in enhanced biodegradation of MBC in soil. Eur J Haematol, 1990 Jan, 44(1), 9 - 17 Characterization of a new malignant human T-cell line (PFI-285) sensitive to ascorbic acid; Helgestad J et al.; A new malignant human T-cell line-labelled PFI-285-has been isolated from a boy with malignant lymphoma . Morphologically, the cells had characteristics of malignant lymphoid cells . The cells presented surface antigens as early cortical lymphocytes and proliferated non-adherently as single cells, independent of T-cell growth factor (IL-2), in liquid culture . The cells had undetectable levels of receptors for IL-2, were not clonogenic in soft agar, but did form tumors in nude mice . Their establishment and continuous growth in vitro was dependent on the number of cells inoculated and on the growth medium used . Cytogenetic alteration, HTLV-1 or reverse transcriptase activity were not detected . The production of known T-cell derived lymphokines such as IL-2, B-cell growth factor(s), alpha-interferon or granulocyte/macrophage colony stimulating or inhibiting factor(s) was not detected . The cells had 5-8% natural killer (NK)-cell activity against NK-cell sensitive target cells (K562) and were not sensitive for NK cells . A most unusual characteristic was the pronounced sensitivity of the cells to ascorbic acid . Concentrations down to 50 mumol/l killed the cells within hours. Life Sci, 1990, 46(7), 489 - 95 The products of the mdr1a and mdr1b genes from multidrug resistant murine cells have similar degradation rates; Cohen D et al.; Two vinblastine-resistant sublines of the murine macrophage-like cell line J774.2, J7.V1-1 and J7.V3-1, overproduce unique forms of P-glycoprotein that are encoded by distinct mdr genes, mdr1b and mdr1a, respectively . Degradation rates of the two P-glycoprotein isoforms were measured by immunoprecipitation of P-glycoprotein . The half-life of immunoprecipitable P-glycoprotein from J7.V1-1 cells was 16.8 +/- 0.5 hours and from J7.V3-1 cells, 17.4 +/- 0.5 hours . This rate was not influenced by the presence of vinblastine in the growth medium . The data indicate that P-glycoproteins derived from distinct genes have similar degradation rates. Am J Physiol, 1990 Jan, 258(1 Pt 2), R198 - 204 Counteracting effects of urea and betaine in mammalian cells in culture; Yancey PH et al.; Urea and methylamines, such as betaine, are among the major organic osmotic effectors accumulated by organisms under hyperosmotic (high NaCl) stress; the mammalian renal medulla also accumulates such compounds in antidiuresis . Studies on isolated proteins show that urea generally destabilizes protein structure, whereas methylamines are generally stabilizers capable of offsetting the effects of urea . The counteracting-osmolytes hypothesis predicts that cells exposed to high urea concentrations require methylamines for optimal function . In this study, urea, betaine, and other solutes (NaCl, glycerol, sorbitol) were added to growth medium of cultured mammalian cells under conditions in which most solutes entered the cells . For two renal {Madin-Darby canine kidney (MDCK) and PAP-HT25} and one nonrenal (Chinese hamster ovary) cell line, urea (greater than 100 mM) or betaine (greater than 50-100 mM) alone inhibited cell growth and survival, measured as colony-forming efficiency . However, the addition of betaine (up to 120 mM) to media with urea (50-300 mM) greatly increased colony-forming efficiency above that with urea alone . A similar, although less marked effect, was seen on colony sizes with MDCK cells . These results support the counteracting-osmolytes hypothesis. Life Sci, 1990, 46(2), 91 - 8 Opioid-dependent growth of glial cultures: suppression of astrocyte DNA synthesis by met-enkephalin; Stiene-Martin A et al.; The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined . Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media (controls), 1 microM met-enkephalin, 1 microM met-enkephalin plus the opioid antagonist naloxone (3 microM), or naloxone alone (3 microM) . Absolute numbers of neural cells were counted in unstained preparations, while combined {3H}-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes . When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in {3H}-thymidine incorporation by GFAP-positive cells with flat morphology . These results indicate that met-enkephalin suppresses astrocyte growth in culture. J Cell Physiol, 1990 Jan, 142(1), 15 - 20 Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro; Ethier SP et al.; We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures . RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration . Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity . The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range . Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM . Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone . Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations . Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor . These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium . Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner. J Bacteriol, 1990 Jan, 172(1), 149 - 54 Osmoregulation in Rhodobacter sphaeroides; Abee T et al.; Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light . The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths . The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions . Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized . The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM) . Only one kinetically distinguishable choline transport system could be detected . Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively . Choline transport was not inhibited by a 25-fold excess of L-proline or betaine . Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl . In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein . This system was also able to transport L-proline, but with a lower affinity than that for betaine . The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems. J Virol, 1990 Jan, 64(1), 458 - 62 Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production; Guetta E et al.; Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice {(parvovirus MVM(p)}, became sensitive to MVM when transformed by polyomavirus . This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus . The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection . The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones . Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used. Arch Microbiol, 1990, 154(4), 317 - 22 Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat; Greer CW et al.; A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source . BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml . Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium . The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage . Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration . BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release. Tsitologiia, 1990, 32(4), 364 - 70 {The cultivation of hybridoma cells in agarose granules}; Kukhareva LV et al.; The results of cultivation of agarose gel immobilized hybridoma cells producing monoclonal antibodies to human alpha 2-interferon are presented . The immobilized cultured cells retained the higher viability for 10-28 days without changing the growth medium, in comparison with cultured cells in control suspension . The cells immobilized in agarose gel demonstrated a tendency to polyploidization as was revealed by the technique of DNA cytofluorimetry with Hoechst 33258. Mol Gen Genet, 1990 Jan, 220(2), 283 - 8 Regulation of chorismate mutase in Saccharomyces cerevisiae; Brown JF et al.; The Saccharomyces cerevisiae ARO7 gene was cloned by screening a wild-type gene bank for complementation of an aro7 auxotrophic mutant . In vitro mutagenesis of the isolated plasmid (pJFB1) gave several transformants resistant to levels of the phenylalanine analogue 2-thienylalanine inhibitory to the wild-type transformant . Chorismate mutase assays indicated that two of the mutants (J14-26IV6 and J14-26IV9) were resistant to feedback inhibition by tyrosine displayed by wild-type strains . Analysis of the effect of other aromatic amino acids on chorismate mutase activity showed that tryptophan counteracted this inhibition . Analysis of the effect of tyrosine in the growth medium on enzyme activity indicated that the wild-type ARO7 gene was repressed by tyrosine, a phenomenon not previously reported . Two of the 2-thienylalanine resistant mutants (J14-26IV3 and J14-26IV9) appeared to be resistant to this repression . Transcriptional analysis confirmed that the level of ARO7 transcript decreased with increasing tyrosine concentration . In stain J14-26IV9 the ARO7 transcript level was not affected . J14-26IV9, therefore, appears to be a double mutant, resistant to both feedback inhibition and repression by tyrosine. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 51 - 3 Mutants of Aspergillus nidulans able to grow at extremely acidic pH acidify the medium less than wild type when grown at more moderate pH; Rossi A et al.; Mutations conferring the ability to grow on extremely acidic media have been selected in the fungus Aspergillus nidulans and map to at least four genes . The mutations fall into two classes: those that confer acid resistance in media of both high and low buffering capacity and those that confer resistance only in media of low buffering capacity . In growth media of more moderate pH mutations of both classes result in reduced acidification of the medium. Ciba Found Symp, 1990, 148, 145 - 55; discussion 155-7 Contributions of autocrine and non-autocrine mechanisms to tumorigenicity in a murine model for leukaemia; Dunn AR et al.; We have surveyed the possible mechanisms by which factor-dependent FDC-P1 cells can be rendered leukaemogenic by exposure of cells to the chemical mutagen, ethyl methane sulphonate . Cell lines established on the basis of an ability to proliferate in the absence of exogenous colony-stimulating factors (CSFs) fall into two classes; those that are maximally stimulated and show no evidence of production of CSFs and others that grow in a density-dependent manner and express granulocyte-macrophage CSF (GM-CSF) . That the growth of this latter class can be suppressed by the inclusion of antisense GM-CSF oligonucleotides in the growth medium indicates that the basis for their in vitro proliferation, and probably their ability to initiate the formation of transplantable leukaemias, is autocrine stimulation by GM-CSF . The ability of low levels of CSF to sustain autocrine stimulation, as we have shown, raises the possibility of an autocrine basis for the proliferation of certain human leukaemic cells . The ability to detect low concentrations of CSFs and develop in vitro assays that closely mimic the conditions that exist in vivo will be important aids in the classification of human leukaemias. Int J Dev Neurosci, 1990, 8(4), 361 - 70 The effects of potassium-induced depolarization, glutamate receptor antagonists and N-methyl-D-aspartate on neuronal survival in cultured neocortex explants; Ruijter JM et al.; The effects of elevating the potassium concentration of the growth medium of neocortical explants was studied . Under control conditions, 10 mM potassium resulted in ca 20% decrease in the number of surviving neurons . The same potassium concentration, however, was clearly neurotrophic in tetrodotoxin-grown cultures: tetrodotoxin-induced neuronal death was significantly reduced . Both effects could be mimicked by the addition of 10 microM N-methyl-D-aspartate (NMDA); lower concentrations were without effect; higher concentrations were neurotoxic under both control and tetrodotoxin conditions . The neurotoxic, as well as the neurotrophic effects of 10 mM potassium appear to be mediated through depolarization-induced glutamate release since they could be influenced by the application of glutamate receptor antagonists . The addition of the NMDA receptor antagonist D-2-amino-7-phosphonoheptanoate (APH) blocked the trophic effect of 10 mM potassium in tetrodotoxin-grown cultures, resulting in low survival . On the other hand, the addition of the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) resulted in neuronal survival similar to control cultures, indicating that it blocked the toxic effects of glutamate, leaving the trophic effects on the NMDA receptor untouched . Under control (non-TTX) conditions, neither DNQX nor APH showed significant effects on 10 mM potassium-induced cell death, indicating that stimulation of the non-NMDA, as well as the NMDA receptors is neurotoxic . This differential effect of NMDA receptor stimulation on neuronal survival is discussed with respect to the maturational and/or functional state of the neurons in the culture. Comp Biochem Physiol B, 1990, 96(4), 775 - 82 Effect of mitochondrial inhibitors on adenosinetriphosphate levels in Plasmodium falciparum; Fry M et al.; 1 . The effects of mitochondrial inhibitors on the ATP levels of intraerythrocytic Plasmodium falciparum have been studied . 2 . Changes in parasite ATP or ADP levels with time in response to various mitochondrial inhibitors appear quite complex; ATP levels may be initially depressed and then elevated above normal, but the nature of the response depends upon the stage in the intraerythrocytic cycle and in some cases upon the concentration of the inhibitor used . 3 . After ca 2 hr incubation of cultures with inhibitors ATP levels appear to be stabilized and are similar to those of untreated parasites . However, ADP levels of trophozoites show significant increases after a 2 hr incubation with inhibitors, particularly with oligomycin and to a lesser extent with antimycin A; increases in ADP levels however were not observed in ring-stages of the parasite . 4 . Inhibition of red cell and parasite glycolysis leads to rapid decreases in parasite ATP levels which are not significantly affected by oligomycin . Incubation of in vitro cultures with oligomycin can result in a decreased, rather than increased rate of lactate production with a concomitant appearance of pyruvate in the growth medium . 5 . This investigation would indicate that if there is a mitochondrial contribution to the parasite ATP pool it is relatively small, and that a short-fall in this contribution is quickly compensated for by ATP from other source(s), although this is not necessarily met by increased glycolysis. Biomed Biochim Acta, 1990, 49(4), 289 - 91 Influence of hydrazinophthalazines on the activity of collagenase in murine fibroblast culture; Weglarz L et al.; Murine fibroblasts cultured in vitro were treated with hydrazinophthalazine drugs inducing the lupus erythematosus-like syndrome . Collagenase enzyme activity in the growth medium was found to be increased 3-fold by hydralazine and 1.8-fold by binazine, as compared to the control cultures . The enhanced level of collagenolytic activity points to the metabolic changes in the connective tissue in response to hydrazinophtalazines. Annu Rev Nutr, 1990, 10, 319 - 35 Folate-binding proteins; Henderson GB; Folate-binding proteins of three major classes have been observed in various bodily fluids and in the plasma membrane and cytoplasm of normal and neoplastic cells . A major class, the high-affinity folate-binding proteins, show a preferential and tight binding of folic acid relative to reduced folates and methotrexate and consist of water-soluble and membrane-associated forms . Soluble forms of the high-affinity binders are present in serum and milk and in the growth medium of certain cultured cell lines, whereas membrane-associated forms are observed on the surface of various cells and tissues . The binders in serum have no clearly defined function, whereas the milk binders serve to accumulate and stabilize reduced-folate compounds in milk and they may also facilitate the absorption of folates by the intestinal mucosa of neonates . Membrane-bound forms of high-affinity folate-binding proteins mediate the transport of folate compounds across plasma membranes and appear to utilize endocytosis as the transport mechanism . Membrane-associated high-affinity binding proteins contain covalently bound phospholipids and hydrophobic C-terminal amino acid sequences that are absent in the soluble forms . The remaining protein portions of these binders show considerable sequence homology . The second class is composed of folate-binding proteins that reside solely in the plasma membrane and are structurally and mechanistically distinct from the high-affinity binders . These proteins function in transport, exhibit varied substrate specificities that accommodate reduced-folate compounds with equal or higher affinity than folate, and do not utilize endocytosis as the mechanism for substrate internalization . The third class of folate-binding proteins consists of enzymes that reside in the cytoplasm of cells. J Gen Microbiol, 1990 Jan, 136 ( Pt 1), 137 - 46 Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii; Vandercammen A et al.; Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min . The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period . All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose . In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination . Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose . The remaining glycogen was degraded upon subsequent germination of the zoospores . B . emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein . On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved . Upon incubation of fractions from the two peaks or of a crude extract in the presence of {2-32P}fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained . The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column . PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained . Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B . emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate . The physiological significance of these properties is discussed . A simple method for the determination of trehalose is also reported. Antimicrob Agents Chemother, 1990 Jan, 34(1), 111 - 6 Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system; Chopra I et al.; A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing a cloned tetA-lacZ gene fusion, is described . Expression of beta-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of beta-galactosidase synthesis . Because synthesis of beta-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay . All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium . A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed. Int J Hyperthermia, 1990 Jan-Feb, 6(1), 33 - 46 Possible involvement of ubiquitin function and ATP requirement in the development of thermotolerance in mammalian cells; Mizuno S et al.; Thermotolerance under chronic exposure to moderate hyperthermia at 41 degrees C was hardly induced in the mouse temperature-sensitive mutant ts85 cells, in contrast to the parental wild-type FM3A cells . Thermotolerance was induced at a reduced level in the mutant cells compared with the wild-type cells by incubation at 33 degrees C (permissive temperature), but not at 39 degrees C (non-permissive temperature), after a brief exposure at 44 degrees C . Under conditions where protein synthesis was inhibited by cycloheximide at 41 degrees C, significant amounts of thermotolerance developed in FM3A cells . FM3A cells depleted of cellular ATP by treatment with 2.4-dinitrophenol and 2-deoxyglucose were not sensitized to thermal cell killing at 44 degrees C, when drug-treated cells were washed and exposed to hyperthermia in drug-free growth medium, where cellular ATP rapidly recovered . However, the cells deprived of ATP under the treatment at 41 degrees C failed to develop thermotolerance, indicating a requirement of ATP for thermotolerance development . The decay of thermotolerance was not affected by ATP levels after it was developed . The degradation of abnormal cellular proteins which contained amino acid analogues was promoted at 33 degrees C relative to normal protein degradation in FM3A and ts85 cells . Both normal and abnormal proteins were degraded at a reduced rate at 43 degrees C . Pretreatment of cells at 41 degrees C decreased the rate of degradation of abnormal proteins at 33 degrees C by 20% in FM3A cells and by about 100% in ts85 cells . Pretreatment of cells at 41 degrees C increased significantly the conjugation of 125I-labeled ubiquitin to cellular endogenous proteins in extracts of FM3A cells, but decreased the conjugation in extracts of ts85 cells . The data presented here, in conjunction with the observations by others that the ts85 cell is a mutant defective in the ubiquitination of cellular proteins at nonpermissive temperatures, suggest that the ATP-dependent ubiquitination may be crucial for the development of thermotolerance. Haemostasis, 1990, 20(6), 313 - 20 Female sex hormones and platelet/endothelial cell interactions; Berge LN et al.; The effects of estradiol and progesterone added to the growth medium of human umbilical vein endothelial cells for 72 h on the formation and release of prostacyclin were investigated . The influence on collagen-induced platelet aggregation and on the platelet formation of thromboxane A2 following aggregation, of the growth medium collected before and after thrombin stimulation of the endothelial cells, was studied simultaneously . Under basal conditions, endothelial cells grown with progesterone released significantly less prostacyclin into the growth medium than did controls (p less than 0.05) . Following thrombin stimulation, endothelial cells grown with estradiol (p less than 0.05) or a combination of estradiol and progesterone (p less than 0.01) contained significantly less prostacyclin than controls . No significant effects on the platelet aggregation or platelet thromboxane formation could be found . This study indicates a lowering effect of both female sex hormones on the endothelial cell prostacyclin formation and release . This may be of significance for the increased risk of vascular disease in pregnant women and oral contraceptive users, but can hardly explain the consequences of the hormonal loss occurring at the menopause. Dev Genet, 1990, 11(5-6), 403 - 9 Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells; Knecht DA et al.; A new method for clonal growth of Dictyostelium axenic amoebae has been developed . Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose . Cells grow normally in the agarose and form colonies up to several millimeters in diameter . When the colonies have grown to a sufficient size, they begin multicellular development . Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation . Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies . This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important . This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation . Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified . Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation . The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells. Arch Microbiol, 1990, 155(1), 7 - 12 Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli; Heitzer A et al.; Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions . Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion . During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition . However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content . During heat shock, tight temperature controlled expression was evident . While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any given dilution rate . From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used. Arch Oral Biol, 1990, 35(12), 967 - 76 Characterization of human gingival keratinocytes cultured in a serum-free medium; Wille JJ et al.; Primary cultures of keratinocytes were established from gingival tissue explanted on the surface of type I collagen gels and fed a serum-containing medium . Cells could be routinely subcultured for at least five passages in a basal nutrient medium (MCDB 153) containing low calcium (0.1 mM), and supplemented with ethanolamine, phosphoethanolamine, hydrocortisone, insulin, epidermal growth factor and protein of bovine pituitary extract . Cells seeded at low densities doubled exponentially in number every 24-30 h and formed a confluent monolayer within 10-14 days . Phase-contrast light and transmission electron microscopy showed that the keratinocyte cultures had features typical of epithelial cells, including desmosomes and perinuclear tonofilament bundles . Immunofluorescent microscopy showed the presence of specific keratin proteins in basal cells of proliferating cultures . Gel electrophoresis of the insoluble cytosolic proteins of gingival and skin keratinocytes showed several differences . Suspension of dividing gingival keratinocytes in 1.3% methylcellulose medium induced greater than 50% cross-linked envelopes, suggesting the existence of a terminal differentiation pathway in gingival basal cells . Clonal growth experiments showed that both insulin and epidermal growth factor were required for optimal clonal growth . The growth of subcultures was arrested and the unstratified epithelial monolayer induced to form a stratified sheet by replacing the growth medium with basal MCDB 153 medium depleted of growth factors and containing 2 mM calcium . Sheets of stratified gingival epithelium formed on and later released from the dish by enzymatic treatment may be suitable for a variety of experimental and clinical uses. Free Radic Res Commun, 1990, 11(1-3), 65 - 76 Oxidative stress and tumour cell proliferation; Burdon RH et al.; The effects of oxidant stress were studied in immortalised hamster (BHK-21) and rat (208F) cell lines before and after transformation to the malignant state with polyoma virus, or activated H-ras, respectively . Whilst intracellular superoxide production was detectable in both transformed and immortalised cells the rate was somewhat higher in the transformed cells which have lower levels of superoxide dismutase . Because growth of transformed cells was particularly depressed in the presence of MTT, a tetrazolium compound reduced by superoxide, the possible role of active oxygen species in the promotion of cell growth was examined . Low levels of hydrogen peroxide were stimulatory towards both immortalised and transformed cells . In the case of H-ras transformed rat cells, paraquat was also stimulatory provided serum was present in the growth medium . In the absence of serum, paraquat was notably inhibitory but inhibition could be alleviated by addition of low concentrations of alpha-tocopherol (10(-8)M) to the serum-depleted medium . Although depletion of serum from the growth medium also leads to lower cell proliferation, subsequent experiments showed that alpha-tocopherol addition to serum-free medium was sufficient to restimulate growth . In the case of transformed cells, yields of cells were even greater than that encountered in the presence of 10% serum . Thus whilst certain active oxygen species (e.g . hydrogen peroxide) may have a role in promoting the growth of transformed and immortalised cells the necessity for antioxidant protection is important. Cytotechnology, 1990 Jan, 3(1), 61 - 73 Long-term culture of human esophageal explants and cells; Resau JH et al.; Human esophageal epithelium obtained from intermediate autopsies (less than 12 h) was maintained as cell and explant cultures . In order to develop a serum-free, defined media culture model, several medias and additives were evaluated . The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066 . The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased 3H-TdR labelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM) . The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations . The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells . Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins . Explants cultured in these dishes were equally well-preserved and differentiated . There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating . A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells . Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells. J Ind Microbiol, 1990 Jan, 5(1), 9 - 15 Characterization of the requirements and substrates for reductive dehalogenation by strain DCB-1; Linkfield TG et al.; An obligately anaerobic bacterium known as strain DCB-1 was grown under a variety of conditions to determine the requirements for dehalogenation as well as factors which stimulated or inhibited the process . Dechlorination was obligately anaerobic since introduction of O2 immediately inhibited the reaction . Sulfuroxy anions, which also serve as electron acceptors for DCB-1, inhibited dechlorination but NO3- and fumarate did not . The optimum growth medium for dechlorination was 0.2% Na pyruvate and 20% rumen fluid in basal salts . Media with either pyruvate or rumen fluid alone did not support dechlorination . DCB-1 also consumed H2 but typical substrate concentrations of H2 (80 kPa) delayed dechlorination . Once the H2 concentration was reduced to less than 20 microM (2.67 kPa), dechlorination resumed . Dehalogenation by DCB-1 was restricted to the meta substituted benzoates as halogens in other positions and chloroaromatic compounds with other functional groups were not dechlorinated. Mol Microbiol, 1989 Dec, 3(12), 1709 - 18 Nickel deficiency gives rise to the defective hydrogenase phenotype of hydC and fnr mutants in Escherichia coli; Wu LF et al.; Hydrogenase activity and other hydrogenase-related functions can be restored to hydC mutants by the specific addition of nickel salts to the growth medium . These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis . The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain . Partial suppression of the hydrogenase phenotype of hydC mutants occurs when growth takes place in a different medium . This correlates with an increased cellular nickel content . The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content . Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content . Mutations in the fnr gene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolishes hydC expression and gives rise to a defective hydrogenase phenotype . The hydrogenase phenotype of fnr is closely similar to that of hydC in all respects examined . The hydrogenase activity of fnr strains can be restored by the presence of a functional hydC gene on a multicopy plasmid . The hydrogenase phenotype of fnr strains therefore arises indirectly via suppression of hydC, which leads to a low cellular nickel content . Nickel has no influence on fumarate reductase or nitrate reductase activities in fnr strains . The hydrogen-metabolism phenotype of fnr strains is, therefore, dependent upon their ability to acquire nickel from growth media . It is likely that hydC encodes a specific transport system for nickel. Mol Cell Biol, 1989 Dec, 9(12), 5516 - 24 Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1; Chambers A et al.; The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG . The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3' . The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF . We have demonstrated that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1 . Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium . PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate . This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence . The promoters of many other yeast glycolytic genes also contain consensus RAP1-binding sites and copies of the CTTCC pentamer . This suggests that RAP1 may be involved in transcriptional control of many other glycolytic genes in addition to the PGK gene. J Cell Physiol, 1989 Dec, 141(3), 483 - 9 Different pattern of differentiation in two LLC-PK1 clones; Van den Bosch L et al.; Two clones (LD3 and LC3) were isolated from the established renal cell line LLC-PK1 . They differed with respect to the development of the Na+-dependent alpha-methyl-D-glucoside (AMG) uptake . The higher uptake capacity in LD3 as compared to LC3 was owing to the expression of a higher number of carrier molecules as was shown by the specific phlorizin binding . The intracellular cyclic AMP level, the D-glucose concentration in the growth medium and the cell density could be excluded as the causes of the difference between both clones . We found that the faster development of the Na+-dependent hexose carrier in LD3 was parallelled by a faster expression of trehalase in this clone . This suggests that the expression of both apical proteins is correlated . From the growth curves it was concluded that renewal of the undifferentiated population was roughly the same in both clones . The recruitment of cells from the undifferentiated to the growth arrested state seems also very similar as was deduced from the development of tight junctions and from the down-regulation of the alpha-methylaminoisobutyric acid (meAIB) uptake . The start of differentiation was identical as was shown by the similar rate of expression found for gamma-glutamyl transferase . The difference between both clones is most likely situated at the traverse to a fully differentiated cell . This process takes more time in LC3 than in LD3 . Also the fully differentiated state seemed to be different in both clones . We conclude that the pattern of differentiated characteristics found in both clones is different and a model incorporating these differences is presented. J Gen Microbiol, 1989 Dec, 135 ( Pt 12), 3421 - 9 Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat; van der Woude MW et al.; The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates . The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu) . Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1) . Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1) . Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu . The composition of the growth medium influenced both phase variation and overall production of fimbriae . A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells . A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell . The frequency of the phase switch was calculated for different growth conditions . The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2) . In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2) . The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control. J Gen Microbiol, 1989 Dec, 135 ( Pt 12), 3311 - 8 Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae; Vancurova I et al.; Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP . The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium . NADP-dependent GDH was purified to homogeneity from crude extracts of S . fradiae . The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer . The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor . The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate . The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP . Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4 . The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1989 Dec, 11(6), 395 - 401 {Abnormal inositol phospholipid metabolism as a main factor causing pericyte drop-out in diabetic retinopathy}; Li WY; The biochemical basis of pericyte loss in early diabetic retinopathy is still an open question . In studies on the mechanism by which retinal pericytes degenerate, we first established a bovine retinal capillary pericyte (BRCP) cell line . Subcultured BRCP grown in high (10-40 mmol/L) glucose media were used as an experimental model . We found that high concentrations of glucose suppress the mitotic rate and cell birth rate of cultured BRCP . High concentrations of glucose inhibit myo-inositol transport and result in decreased intracellular myo-inositol content . This inhibition can be partially reversed by sorbinil, an aldose reductase inhibitor (ARI) . Myo-inositol is a precursor of inositol phospholipids (IPLs), whose metabolism is responsible for a number of signal transduction processes . Phosphoinositidase (PIase) cleaves the phosphodiester bond of phosphotidylinositol 4,5 diphosphate (PIP2) to produce two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG) . Further experiments showed that IP3 and DG synergistically activate BRCP proliferation in vitro . High concentrations of glucose altered the formation of both IPLs and inositol phosphate esters (IPEs) in an organ culture of retinal microvessels . This alteration can be reversed by adding either high concentrations of myo-inositol or ARI to the medium . PIase activity was attenuated to 82% or 55% when glucose in the growth medium was increased from 5 to 15 or 30 mmol/L, respectively . When IPLs from BRCP were analyzed by HPLC and TLC, we observed the reduction of three IPLs, including the substrate of PIase, PIP2.(ABSTRACT TRUNCATED AT 250 WORDS) Indian J Biochem Biophys, 1989 Dec, 26(6), 367 - 70 Further studies on the influence of dibutyryl cAMP, theophylline and prostaglandin E1 on composition and biosynthesis of phospholipids in Microsporum gypseum; Vaidya S et al.; Exogenous supplementation of dibutyryl cAMP and cAMP modulators like theophylline and prostaglandin E1 in the growth medium of Microsporum gypseum lead to increase in the levels of phosphatidylcholine and lysophosphatidylcholine and thereby in total phospholipid content . These observations were further confirmed by the increased incorporation of {32P}orthophosphoric acid into total phospholipid and {14C}choline into phosphatidylcholine and lysophosphatidylcholine . The activity of sn-glycerol-3-phosphate acyltransferase, the enzyme involved in phospholipid synthesis, was stimulated in the presence of dibutyryl cAMP, theophylline and PGE1 supporting the increased synthesis of phospholipids. Biochim Biophys Acta, 1989 Nov 20, 1014(2), 162 - 72 Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol; Tang BL et al.; The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium . The enhancement was not due to increased bioavailability because cellular uptake of {3H}tamoxifen was not increased and the lipoprotein fraction of serum had negligible {3H}tamoxifen-binding capacity . Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens . Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins . The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce {3H}thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce {3H}tamoxifen binding by 50%) (correlation coefficient 0.94) . Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol . These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells. FEBS Lett, 1989 Nov 20, 258(1), 123 - 6 Expression of catalytically active rat S-adenosylmethionine decarboxylase in Escherichia coli; Salmikangas P et al.; The cDNA coding for rat S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) has been cloned into a plasmid expression vector, pKK-223-3, and expressed in E . coli . The authenticity of the expressed protein has been demonstrated by reactivity with antibodies specific for rat AdoMetDC, by size analysis on SDS gels visualized with immunotransblots, and, finally, by catalytic activity . The expression of the enzyme results in a decrease in the activity of the bacterial enzyme suggesting the replacement of the bacterial enzyme by the rat AdoMetDC . Similarly, the addition of exogenous spermidine to the growth medium reduces bacterial enzyme activity affecting only marginally the expression of the recombinant protein. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1331 - 8 Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene; Vanoni M et al.; The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins . We have used the signal sequence from the S . diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene . In frame STA2/lacZ gene fusions have been constructed and expressed in S . cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters . Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space . Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase. Eur J Biochem, 1989 Nov 6, 185(2), 469 - 74 Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent; Rosenberg-Hasson Y et al.; Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines in mammalian cells is characterized by an extremely short half-life . In the present study, ODC degradation was investigated in 653-1 mouse myeloma cells that overproduce ODC and in ts85 cells that are thermosensitive for conjunction of ubiquitin to target proteins . Addition of 2-deoxyglucose and dinitrophenol (agents that efficiently deplete cellular ATP) to the growth medium of these cells inhibited ODC degradation . In contrast, chloroquine and leupeptin, inhibitors of intralysosomal proteolysis, did not affect ODC degradation . Shifting ts85 cells to 42 degrees C (a non-permissive temperature that inhibited conjugation of ubiquitin to target proteins) did not prevent ODC degradation . The ATP-dependent degradation of ODC in 653-1 cells was inhibited substantially by N alpha-tosyl-L-lysine chloromethane (TosPheMeCl), iodoacetamide and o-phenanthroline . These results suggest that ODC degradation occurs via a non-lysosomal . ATP-requiring and ubiquitin-independent cellular proteolytic mechanism, and that serine proteases and enzymes containing sulphydryl groups and metalloenzyme(s) may be involved in this process. Anticancer Res, 1989 Nov-Dec, 9(6), 1611 - 5 Growth kinetic studies of methionine dependence in co-culture of monolayer and anchorage independent mouse cell lines; Djurhuus R et al.; Non-transformed C3H/10T1/2 Cl 8 mouse embryo fibroblasts and malignant R.1.1 mouse T-lymphoma cells were examined for their ability to utilize homocysteine thiolactone (Hcy-tl) instead of methionine (Met) for growth . The non-transformed fibroblasts showed only a slightly slower growth rate in Hcy-tl supplemented medium, while the T-lymphoma cells showed an absolute requirement for Met, defined as methionine dependence . A co-culture system was established where both monolayer growing fibroblasts and lymphoma cells in suspension were grown in the same culture vessel . In Met supplemented medium both cell types proliferated, but when Hcy-tl replaced Met only the fibroblasts were able to grow . Two major conclusions were drawn: 1) The inability of the lymphoma cells to utilize Hcy-tl was not due to formation and release of toxic agent(s) to the growth medium . 2) It was possible to exploit the metabolic defect of methionine dependence to select for the growth of non-transformed cells from malignant cells. J Invertebr Pathol, 1989 Nov, 54(3), 306 - 13 The extracellular acid phosphatase of the mosquito-parasitizing fungus Lagenidium giganteum; Bell TJ et al.; The mosquito-parasitizing fungus Lagenidium giganteum secreted a soluble acid phosphatase and beta-D-glucosidase into the growth medium . The acid phosphatase was isolated and purified to single component, and some of its physicochemical properties were determined . The enzyme exhibited a pH optimum of 5.6 in phthalate buffer with p-nitrophenyl phosphate and was temperature-inactivated at 55 degrees C . Enzyme activity seems to be limited to phenyl-phosphate substrates . A molecular weight of 42,800 was found and the amino acid content was also determined . A Km for p-nitrophenyl phosphate of 1.6 x 10(-7) M was found . The possible involvement of the enzyme in the infective process was discussed. J Endocrinol, 1989 Nov, 123(2), 233 - 41 Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium; Chaminadas G et al.; Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated . Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium . For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells . Only experiments in which the control dishes displayed more than 80% of anticytokeratin-immunostained cells were further processed . After this period oestradiol-17 beta (20 nmol/l; control), oestradiol-17 beta (20 nmol/l) plus progesterone (0.5 mumol/l), oestrone sulphate (1 mumol/l) or oestrone sulphate (1 mumol/l) plus progesterone (0.5 mumol/l) were added to the medium for 48 h . An immunocytochemical progesterone receptor assay showed that oestradiol-17 beta increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17 beta induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells . In the