Growth media

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 these culture conditions and after 16 h of incubation, oestradiol-17 beta induced a 1.7-fold increase in {3H}thymidine incorporation into DNA, and {35S}methionine incorporation into cellular proteins was linearly increased up to 8 h . Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of {35S}methionine . The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1989 Nov 1, 143(9), 2850 - 7
Hodgkin's cell lectin, a lymphocyte adhesion molecule and mitogen; Paietta E et al.; We have previously shown a novel galactose/N-acetylgalactosamine specific lectin activity (Hodgkin's disease (HD) lectin) on the surface of cultured HD cells (lines L428, its variants, and line L540) to mediate lymphocyte adhesion . We here demonstrate that both surface membrane-bound and secreted HD lectin activities participate in the activation of agglutinated lymphocytes . Among known adhesion molecules expressed by the HD cells, only the intercellular adhesion molecule-1 (ICAM-1) contributed to this activation as an alternative PBL binding site . As yet we have not identified the cellular ligand(s) for the HD lectin on the lymphocyte surface . Pretreatment of lymphocytes with mAb to the accessory molecules CD2, CD3, CD4, CD8, CD11b, or CD11c did not interfere with their response to HD cells . mAb to CD11a (LFA-1), the alleged ligand of ICAM-1, inhibited the ICAM-1 but not the HD lectin-mediated lymphocyte stimulation . Although lymphocyte binding could proceed via either pathway, lymphocyte activation always depended upon factors secreted by the HD cells, one of which we identified as a soluble form of the HD lectin based on its shared properties with the membrane-bound form including immunologic cross-recognition and carbohydrate-binding specificity . Although HD cell-conditioned medium alone stimulated lymphocytes, HD cell plasma membranes could compensate for low concentrations of this medium . In addition, resting lymphocytes, normally unresponsive, were triggered into DNA synthesis by growth medium when cocultured with HD cell membranes . The unique functions of the surface-expressed HD lectin and its soluble counterpart as lymphocyte adhesion molecule and mitogen might be physiologically relevant to the severe immunodeficiencies occurring in patients with HD.

Cell Differ Dev, 1989 Nov, 28(2), 105 - 17
Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor; Chou SC et al.; We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation . A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium . Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week . This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month . We report here that the dedifferentiation of P15 occurs in two stages . One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month . After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D) . Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN) . Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day . There was also an increase in the amount of dopa-positive TGN within one day . Melanosomes first appeared after three days and became numerous after one week . Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells . We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Infect Immun, 1989 Nov, 57(11), 3484 - 90
Reversion of the antichlamydial effect of tumor necrosis factor by tryptophan and antibodies to beta interferon; Shemer-Avni Y et al.; Human recombinant tumor necrosis factor-alpha (TNF-alpha) inhibited the growth of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cells . The effect was synergistic with that of gamma interferon (IFN-gamma) . TNF-induced resistance to chlamydiae could be blocked with cycloheximide, suggesting that it involves the function of some induced proteins . Tryptophan degradation was enhanced in the TNF-treated cells and was much further increased when the cells were treated with both TNF and IFN-gamma at concentrations at which IFN-gamma by itself had very little effect . Antibodies to IFN-beta blocked the augmentation of tryptophan degradation by TNF and decreased but did not fully eliminate the antichlamydial effect of TNF . Increased concentration of tryptophan in the growth medium (greater than 100 micrograms/ml) resulted in reversion of the antichlamydial effect of TNF . This study suggests that the inhibition of chlamydial growth by TNF is mediated partly through an autocrine function of IFN-beta which, in synergism with TNF, enhances the activity of a tryptophan-degrading enzyme(s) and partly by some other activities of TNF which can be blocked by tryptophan.

Virology, 1989 Nov, 173(1), 302 - 10
HPV-18 immortalization of human keratinocytes; Kaur P et al.; The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin . Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection . Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture . Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis . In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames . Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins . However, none of the cell lines tested were tumorigenic . The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride . All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally . K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.

J Biol Chem, 1989 Oct 15, 264(29), 17355 - 60
Glutathione-coated cadmium-sulfide crystallites in Candida glabrata; Dameron CT et al.; Cadmium-sulfide crystallites form in the yeast Candida glabrata cultured in the presence of cadmium salts . The particles function to sequester and detoxify intracellular cadmium ions . The crystallites are peptide-coated, but the coating peptide varies with the nutrient conditions of the growth medium . When cultured in rich nutrient broth the yeast forms intracellular CdS particles coated with a mixture of glutathione and the gamma-glutamylcysteine dipeptide . In contrast, cultures in synthetic minimal medium yield particles coated with polymerized gamma EC peptides of general structure (gamma-Glu-Cys)n-Gly . Glutathione/gamma-glutamylcysteine particles exhibit properties analogous to quantum, semiconductor-type crystallites . The optical properties are dependent on particle size, and irradiation results in photoluminescence and photoreduction not observed in bulk CdS mineral . Aerobic irradiation leads to particle decomposition presumably via oxidation of the sulfide ions within the crystallite.

J Biol Chem, 1989 Oct 5, 264(28), 16537 - 44
Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae; Stukey JE et al.; The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase . This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A . A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells . Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene . Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation . Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium . The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium . Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations . During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids . No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation . These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme . 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.

Mutat Res, 1989 Oct, 214(2), 321 - 8
X-ray-induced G2 arrest in ataxia telangiectasia lymphoblastoid cells; Tatsuka M et al.; Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiectasia (AT) lymphoblastoid cells and normal human cells . Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in AT cells than in normal cells as previously reported by other investigators . However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistinguishable from that in normal cells . Moreover, no significant difference in cell viability was noted between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar . These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to more stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there . The possible reasons for this greater accumulation are discussed.

Infect Immun, 1989 Oct, 57(10), 2991 - 7
Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells; Arroyo R et al.; The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated . Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T . vaginalis to recognize and bind to epithelial cells . Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T . vaginalis cytadherence . Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone . Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment . Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T . vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T . vaginalis in the presence and absence of TLCK were identical . Kinetics of TLCK-mediated inhibition of cytadherence of other T . vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286 . Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence . Finally, treatment of T . vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity . These data show for the first time the involvement of T . vaginalis cysteine proteinases in parasite attachment to human epithelial cells . These results have implications for future pharmacologic intervention at a key step in infection.

Cancer Res, 1989 Oct 1, 49(19), 5435 - 42
Induction of cells with a chondrocyte-like phenotype by treatment with 1 alpha,25-dihydroxyvitamin D3 in a human salivary acinar cell line; Azuma M et al.; A clonal cell with an acinar cell phenotype, which was induced by 5-azacytidine treatment of a neoplastic human salivary intercalated duct cell line, was cultivated in the presence of 1 alpha,25-dihydroxyvitamin D3 . Morphological changes occurred; large cells that were polygonal or round in shape and had numerous vacuoles in their cytoplasm appeared in the treated cells, whereas the same concentration of 1 alpha,25-dihydroxyvitamin D3 did not affect the morphology of the parental cells . Major alterations, such as expression of type II collagen, alpha and beta chains of S-100 protein, and sulfated proteoglycans, were observed in these cells with a phenotype similar to chondrocytes . After the removal of 1 alpha,25-dihydroxyvitamin D3 from the culture, the treated cells returned rapidly to the phenotype of the untreated cells . These findings indicate that the reversible differentiation into chondrocyte-like cells of a human salivary acinar cell line occurs in growth medium containing 1 alpha,25-dihydroxyvitamin D3.

Mol Cell Biol, 1989 Oct, 9(10), 4161 - 9
The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation; Mayer SA et al.; CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA . Cytochrome b is the only mitochondrially synthesized component of the respiratory chain complex III . Since the nuclearly encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated . To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly encoded complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a nonfermentable carbon source . Poly(A)+ RNA from derepressed yeast cells was examined by Northern (RNA) analyses with cRNA probes from CBP1 . Both 2.2- and 1.3-kilobase (kb) transcripts were detected . The 1.3-kb mRNA lacked approximately 900 nucleotides of the 3' end of the 2.2-kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence . Northern analyses of RNA isolated from deletion-insertion mutants of CBP1 and from strains that overexpress CBP1 mRNA demonstrated that both mRNAs were transcribed from the CBP1 gene . Furthermore, we demonstrated that the levels of the two CBP1 mRNAs were reciprocally regulated by the carbon source in the growth medium . This is the first description of a yeast gene from which two transcripts that can encode proteins with distinctly different coding properties are generated by alternative 3'-end formation.

J Virol, 1989 Oct, 63(10), 4264 - 76
Varicella-zoster virus glycoprotein oligosaccharides are phosphorylated during posttranslational maturation; Gabel CA et al.; Varicella-zoster virus (VZV)-infected human embryonic lung fibroblasts (HELF) do not release infectious virions into their growth medium . Extracellular virions are pleomorphic, suggesting that they are partially degraded before their release from cells . To examine the intracellular pathway of viral maturation, {2-3H}mannose-labeled virus-encoded glycoproteins were isolated from VZV-infected HELF . Oligosaccharides attached to the glycoproteins were processed to complex-type units, some of which were phosphorylated . The major intracellular site of accumulation of VZV gpI was found to be perinuclear and to correspond to that of the cation-independent mannose 6-phosphate (Man 6-P) receptor . Subsets of VZV-containing cytoplasmic vacuoles were coated, Golgi-associated, or accessible to endocytic tracers . Phosphorylated monosaccharides protected HELF from the cytopathic effect of VZV in proportion to their ability to block Man 6-P receptor-mediated endocytosis . These data suggest that the unusual phosphorylated oligosaccharides mediate an interaction between VZV and Man 6-P receptors of the host cell; this interaction may be responsible for withdrawal of newly synthesized virions from the secretory pathway and for their diversion to prelysosomal structures.

J Clin Endocrinol Metab, 1989 Oct, 69(4), 868 - 74
Estrogen synthetase (aromatase) activity in primary culture of human term placental cells: effects of cell preparation, growth medium, and serum on adenosine 3',5'-monophosphate response; Lobo JO et al.; The establishment of human term trophoblast cells in culture is dependent on the method of cell preparation, growth medium used, and presence of serum . Using freshly isolated term placental cells, we investigated 1) the effects of two different methods of removal of nontrophoblast cells and two culture media on trophoblast aromatase specific activity, cellular morphology, and hCG secretion over 72 h; and 2) the sensitivity of these hormonal parameters to cAMP added 24 h after the initiation of culture . Under conditions in which aromatase is responsive to cAMP, we studied the effect of removing serum after 24 h in culture with serum . After trypsin digestion of placental villi, isolated trophoblast cells were either treated with ammonium chloride (A) or purified on Percoll density gradients (P) and then grown in monolayer culture with medium 199 plus 10% fetal bovine serum (M) or Dulbecco's Modified Eagle's Medium (DMEM) plus 20% fetal bovine serum (D) . Regardless of the method of treatment or growth medium used, aromatase specific activity increased 10- to 15-fold 24 h after plating, continued to increase from 24-48 h, and then decreased (AM) or remained constant (AD, PM, and PD) from 48-72 h . Addition of cAMP significantly increased activity in DMEM-grown cells (PD or AD) at both 48 and 72 h . Aromatase activity in PM cells grown with cAMP increased at 48 h, then decreased to near-control levels by 72 h; however, in AM cells, no response to cAMP was observed relative to control cells . Secretion of hCG was suppressed for the first 48 h in all cultures, but increased by 72 h (greatest increase in AM cultures) . cAMP significantly increased hCG secretion 48 h after its addition under all conditions . We further evaluated the response of the Percoll-purified DMEM-grown cells (PD) to cAMP after serum removal at 24 h . Cells deprived of serum showed significantly higher aromatase specific activity over the entire culture period compared with serum-grown cells . Secretion of hCG increased 2- or 3-fold in the presence or absence of serum, respectively, after 72 h in culture . cAMP increased aromatase specific activity by 1.8- and 1.4-fold at 48 h and by 2.5- and 2.4-fold at 72 h in serum-containing and serum-free cells, respectively . The secretion of hCG increased 11- to 14-fold at 72 h under both serum-containing and serum-free conditions, respectively . The results show that cAMP can act as an intracellular messenger in the regulation of both aromatase activity and hCG secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Exp Cell Res, 1989 Oct, 184(2), 304 - 15
Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans; Morrison P et al.; Heparin is known to bind to cultured endothelial cells . This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern . Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium . The increase is apparent as early as 8 h after heparin administration . Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form . Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion . Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change . These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.

Shika Kiso Igakkai Zasshi, 1989 Oct, 31(5), 603 - 8
Culture conditions for efficient recovery of bacteria from infected dental root canals; Ando N et al.; For better bacterial recovery from endodontic lesions, several bacterial culture conditions were studied . Samples taken from endodontic lesions of 26 freshly extracted teeth, and the bacterial growth was determined in 11 kinds of growth media under aerobic (in air with or without addition of 5% CO2) or anaerobic conditions (in an anaerobic glove box) . Among the media tested, bacteria of endodontic lesions were most efficiently recovered in brain heart infusion medium containing 4% sheep blood (BHI-Blood) under anaerobic conditions . For routine dental practice, serum can be substituted for blood which makes the media opaque, resulting in clear media, and thus bacterial growth can be judged more easily.

J Cell Physiol, 1989 Oct, 141(1), 142 - 7
Possible role of prostaglandins in the regulation of mouse myoblasts; Rossi MJ et al.; A differentiation-defective mouse myoblast subclone (DD-1), cells of which do not fuse into myotubes nor synthesize muscle-specific proteins, was employed to help define the role of eicosanoids in mouse myoblast differentiation . We observed by hplc, tlc, and radioimmunoassay that the DD-1 cells release strikingly higher levels of cyclooxygenase pathway products prostaglandin E2 and F2 alpha into the culture medium than the parental non-differentiation-defective cells (DZ) . In contrast, the levels of 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product, and a putatively identified second lipoxygenase product (LLP) did not differ greatly in the two cell types . The DD-1 cells also have strikingly higher levels of cyclooxygenase activity than the parental cells as determined by intact and broken cell assays . Additional fusion-defective clones were isolated on the basis of their flattened appearance and ability to grow in "mitogen-poor" medium and these cells also released strikingly higher levels of prostaglandins E2 and F2 alpha into the growth medium . The "turn on" of the cyclooxygenase pathway in the DD-1 cells and other fusion-defective cells is consistent with the hypothesis that the products of this pathway contribute to the inability of myoblasts to fuse with one another . This hypothesis is supported by the observation that there is a dose-dependent decrease in fusion of DZ cells when PGE2 is added to commitment medium.

Exp Hematol, 1989 Oct, 17(9), 923 - 8
Effects of beta-D-xylosides on proliferation and matrix formation of adherent fibroblastic cells in mouse bone marrow culture; Nagasaka T et al.; Mouse bone marrow cells were seeded on small pieces of cover glasses placed in culture dishes, and after 3 days, the pieces of glass to which only small spindle cells adhered were transferred to another dish containing fresh medium . The adherent small spindle cells proliferated and some of them became large polygonal cells with abundant cytoplasm . When phenyl beta-D-thioxyloside (0.5 mM), an artificial initiator of chondroitin sulfate chain synthesis, was added to the culture medium, the cells showed a marked increase in number as compared to controls, with conversion of about 35% of the cells to large size cells . Immunohistochemical staining with monoclonal antibodies showed that the intercellular matrix of adherent cells consists mainly of a large proteglylcan with chondroitin 6-sulfate side chains . By histochemical analysis, the amount of chondroitin sulfate was shown to be greater in the intercellular matrices of xyloside-treated groups than those of control cultures . The amount of chondroitin sulfate in the growth medium of the adherent cells, as measured by uronic acid analysis, was also significantly increased by treatment with phenyl beta-D-thioxyloside compared with controls.

J Invest Dermatol, 1989 Oct, 93(4), 449 - 54
All-trans retinoic acid stimulates growth of adult human keratinocytes cultured in growth factor-deficient medium, inhibits production of thrombospondin and fibronectin, and reduces adhesion; Varani J et al.; Human epidermal keratinocytes were established in culture using a low-Ca2+ (0.15 mM), serum-free keratinocyte growth medium (KGM) as the culture medium . Early passage keratinocytes (i.e., between passages 3-8) were incubated for 1 or 2 d in KGM, in KGM supplemented with 1.4 mM Ca2+, or in growth factor-deprived keratinocyte basal medium (KBM) . The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 micrograms/ml), and cell growth was quantitated at the end of the incubation period . The keratinocytes were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN) . Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in KGM . Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca2+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca2+ . In contrast to these effects, all-trans retinoic acid treatment of keratinocytes in KBM, in which the cells were normally quiescent, stimulated growth . In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of keratinocyte proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate) . Keratinocyte adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions . In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid . FN production was also inhibited but to a lesser degree . Concentrations of all-trans retinoic acid required to maximally inhibit keratinocyte adhesion and matrix production were higher (1.0-2.5 microgram/ml) than the concentration required to stimulate proliferation in KBM . These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced keratinocyte proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7453 - 67
A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia; Furfine ES et al.; An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate . One of these species is the previously characterized double-stranded RNA genome of GLV (1) . The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome . This full length single-stranded RNA is not present in viral particles isolated from the growth medium . The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA . The single-stranded species does not appear to be polyadenylated.

Cancer Lett, 1989 Sep 15, 47(1-2), 105 - 10
Estrogens are not required for prolactin induced growth of MCF-7 human breast cancer cells; Vonderhaar BK; MCF-7 human breast cancer cells in continuous culture respond to the growth promoting activity of prolactin . Within 3 days of addition to culture medium in the presence of charcoal stripped fetal bovine serum which is depleted of bovine lactogens and estrogens, prolactin at 250 ng/ml promotes a 2- to 3-fold increase in cell number . When phenol red, which is a weak estrogen agonist, is also eliminated from the medium, the cells respond to prolactin to the same extent . When cells are grown for two generations in the presence of lactogen free, phenol red free, charcoal stripped serum, the prolactin induced response is even greater . Human and ovine prolactins are equipotent . The ability of the cells to bind lactogenic hormones remains unaltered by elimination of phenol red from the growth medium . These data indicate that prolactin alone is a mitogen for human breast cancer cells in long-term culture.

J Biol Chem, 1989 Sep 5, 264(25), 14972 - 7
Alteration of the phospholipid composition of Escherichia coli through genetic manipulation; Heacock PN et al.; In order to study the function of individual phospholipids, we have constructed a strain of Escherichia coli in which the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin can be regulated . In this strain (HDL1001) the normal expression of the phosphatidylglycerophosphate synthase does not occur due to the presence of the pgsA30 allele (Heacock, P . N., and Dowhan, W . (1987) J . Biol . Chem . 262, 13044-13049) . A second chromosomal copy of the pgsA gene is fused to the lacOP region in single copy within the lac operon . Strain HDL1001 is absolutely dependent for growth on an inducer of the lac operon . In addition, the level of the pgsA gene product, the content of the two major acidic phospholipids, and the growth rate are dependent on the level of inducer in the growth medium . Cells remain viable in the absence of inducer as evidenced by a rapid return to normal growth after the readdition of inducer . The growth rate and phospholipid composition are affected only after the level of phosphatidylglycerophosphate synthase drops below about 15% of normal levels; both phosphatidic acid and (d)CDP-diacylglycerol also begin to increase to significant levels . At the point of cell arrest the level of the major acidic phospholipids is reduced by about 90% of wild type levels.

Exp Cell Res, 1989 Sep, 184(1), 53 - 60
A temperature-sensitive mutation in asparaginyl-tRNA synthetase causes cell-cycle arrest in early S phase; Diamond G et al.; The Chinese hamster temperature-sensitive cell-cycle mutant ts24 was analyzed biochemically in order to determine the nature of this lesion . The inability of these cells to proceed through S phase at the restrictive temperature could be complemented by the addition of asparagine to the growth medium, and enzymological analysis showed that this line contains a temperature-sensitive asparaginyl-tRNA synthetase . Normal asparaginyl-tRNA synthetase activity was restored in cells transfected with cloned genomic DNA that overcomes the mutational defect . In corroboration with these results it was shown that a different temperature-sensitive asparaginyl-tRNA synthetase mutant isolated in another laboratory was blocked in S phase in a manner similar to that of ts24 . While the mechanism by which asparaginyl-tRNA synthetase affects cell-cycle progression has not been elucidated, it can be shown that it is not mediated through alteration in overall levels of protein synthesis.

Toxicol Lett, 1989 Sep, 48(3), 265 - 73
V79 Chinese hamster cells express cytochrome P-450 activity after simultaneous exposure to polycyclic aromatic hydrocarbons and aminophylline; Kiefer F et al.; V79 Chinese hamster lung cells expressed low but significant aryl hydrocarbon hydroxylase activities when treated with an inducer of cytochrome P-450I, such as benz{alpha}anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), together with aminophylline . Inducibility by polycyclic aromatic hydrocarbons and inhibition by a specific monoclonal antibody indicated that the observed enzyme activity was mediated by cytochrome P-450I . Intact V79 cells pretreated with TCDD and aminophylline for 24 h metabolized benzo{alpha}pyrene to phenolic products which accumulated linearly in the growth medium for at least the same time period . Exposure of V79 cells to 10 microM benzo{alpha}pyrene and aminophylline for 72 h reduced subsequent cell growth by about 40% . The results demonstrate that V79 cells, under specific conditions, express PAH-inducible cytochrome P-450I and are capable of activating benzo{alpha}pyrene to cytotoxic products.

J Pharmacol Exp Ther, 1989 Sep, 250(3), 1132 - 40
Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line; Miyamoto M et al.; The effects of reducing agents and antioxidants on L-Glutamate (Glu)-induced cytotoxicity were examined in the N18-RE-105 neuronal cell line . The cytotoxicity by Glu (1 and 10mM) was potentiated by exposure to growth medium containing a low concentration of cystine (5-100 microM), instead of the normal medium containing 200 microM cystine . In contrast, the toxicity was suppressed by increasing the cystine concentration to 500 to 1000 microM . Reducing agents, cysteine (30-1000 microM), dithiothreitol (10-250 microM) and glutathione (GSH, 10-1000 microM) also protected the cells against the cytotoxicity of 10 mM Glu in a concentration-dependent manner . The antioxidants vitamin E (10-100 microM), idebenone (0.1-3 microM) and vinpocetine (10-100 microM) also provided marked protection against the cytotoxicity of Glu (10 mM) or quisqualate (1 mM) . Antioxidants also prevented the delayed cell death caused by lowering the concentration of cystine in the medium to 5 microM . Incubation of the cells with 10 mM Glu caused a marked decrease in cellular GSH levels . Although cysteine and dithiothreitol prevented the GSH reduction caused by Glu, antioxidants did not . The cellular levels of oxidants were assessed using 2,7-dichlorofluorescin, a probe that accumulates within cells and is converted to a fluorescent product by oxidation . Glu (10 mM) caused a marked increase in such fluorescence, whereas vitamin E and idebenone reduced markedly the number of fluorescent cells to control levels even added with 10 mM Glu . These results indicate that oxidative stress due to loss of cellular levels of GSH is one mechanism whereby Glu/quisqualate exert cytotoxicity and suggest that centrally active antioxidants may reduce neuronal damage in pathologic conditions associated with excessive Glu release.

Eur J Biochem, 1989 Sep 1, 184(1), 29 - 38
Structural analysis of the carbohydrate chains of a mouse monoclonal IgM antibody; Krotkiewski H et al.; A mouse monoclonal IgM antibody, directed against human blood group B determinant, was isolated from hybridoma culture growth medium . Chemical analysis indicated presence of N- and O-linked oligosaccharides . The N- and O-linked carbohydrate chains were liberated using two different conditions of reductive alkaline degradation . Structural analysis was carried out on the isolated chains using chemical analysis, 500-MHz 1H-NMR spectroscopy and fast-atom-bombardment mass spectrometry . The following composite structures of the N-linked chains were found: (formula; see text) where R = OH for biantennary structures and R = Neu5Ac alpha 2-3Gal beta 1-4 GlcNAc beta 1- or Neu5Ac alpha 2-3Gal beta 1-3{Neu5Ac alpha 2-6}GlcNAc beta 1- for triantennary structures . The O-linked oligosaccharides, found in the light chains, were shown to have the structure Neu5Ac alpha 2-3Gal beta 1-3GalNAc . The native IgM antibody could be separated on a concanavalin-A-Sepharose column into two subfractions, differing in the presence of a high-mannose-type oligosaccharide.

Mikrobiologiia, 1989 Sep-Oct, 58(5), 785 - 90
{The effect of initial bioenergetic state on the cryostability of yeast cells}; Tsutsaeva AA et al.; The cryostability of Saccharomyces cerevisiae cells decreased when they were cultivated under anaerobic conditions in a liquid growth medium YEPD as compared to the culture grown under aerobic conditions . The effect of cultivation conditions on the different cryostability of S . cerevisiae cells is discussed . The initial state of their bioenergetics was shown to influence the cryostability of yeast cells.

J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2413 - 22
The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae; Ramos S et al.; Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium . This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose- or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification . In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described . Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification . Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar . The apparent Km for glucose in this process was 2 mM . Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux . This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol . Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.

Plasmid, 1989 Sep, 22(2), 160 - 2
Amplification of bacterial plasmids without blocking protein biosynthesis; Angelov I et al.; The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied . It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E . coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E . coli plasmids . The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.

Int J Radiat Biol, 1989 Sep, 56(3), 239 - 51
Similarities in the repair kinetics of sublethal and potentially lethal X-ray damage in log phase Chinese hamster V79 cells; Reddy NM et al.; We have compared the repair kinetics of X-ray-induced sublethal damage (SLD) and potentially lethal damage (PLD) in V79 cells during postirradiation incubation in growth medium at 37 degrees C . Kinetics of SLD repair were studied by dose fractionation (D1 + D2 = 2 + 2, 4 + 2, 4 + 4, 10 + 4 and 10 + 7 Gy) . Kinetics of PLD repair were studied by incubating cells with 0.5 M hypertonic saline (HS) for 20 min at various times after irradiation . The repair of SLD, after 2, 4 and 10 Gy, was complete in about 11, 23 and 62 min, respectively . This repair time was independent of the magnitude of dose D2 (test dose) . The repair of PLD, after 2, 4 and 10 Gy, was also complete in about 9, 23 and 61 min, respectively . These data indicate that: (1) the kinetics of repair of SLD for a given dose are independent of the magnitude of the test dose D2; (2) the time taken to complete the repair (repair time) is dependent on dose D1, i.e . the larger the dose D1 the longer the repair time; (3) for a given dose, the repair times assayed either by dose fractionation or by HS treatment are similar; and (4) similarities in the repair times may imply that SLD and PLD are the same . The relationship between the repair kinetics and the shape of the shoulder on fractionated survival curves is discussed.

Mol Pharmacol, 1989 Sep, 36(3), 459 - 64
Growth of S49 cells in low concentrations of beta-adrenergic agonists causes desensitization; Barber R et al.; Epinephrine at concentrations approximating circulating levels in resting subjects produced significant desensitization in wild type S49 lymphoma cells after long term treatment . Desensitization by such low levels of catecholamines was measured by examining subsequent responses of the cells to higher agonist concentrations and was quantified by comparing the integral cAMP accumulations with time in naive and epinephrine-treated cells challenged with the higher epinephrine concentrations . The cells were significantly desensitized after 8 hr of treatment with 3 nM epinephrine or 3 nM terbutaline and were essentially maximally refractory after 24 hr . The 3 nM epinephrine treatment resulted in a small right shift of the EC50 . Responses to epinephrine were partially restored by incubating desensitized cells for 8 hr or longer in growth medium that was free of epinephrine . The attenuation of cAMP responses was largely specific, in that the decrease in the response to prostaglandin was small and the response to forskolin was unchanged . This, together with small increases in cAMP destruction in cell-free preparations from treated cells, suggested that higher phosphodiesterase activity contributed in a minor way to the desensitization . However, the response of the adenylate cyclase system to epinephrine was dramatically attenuated, and very significant changes in the properties of the beta-adrenergic receptors were also obvious . That is, the number of binding sites for epinephrine was reduced by about 65% while the number of sites for {125I}iodocyanopindolol was unchanged . The affinity for the radioactive ligand was significantly reduced . Wild type S49 cells remained viable after several days of continuous treatment with 3 nM epinephrine or terbutaline but responded to subsequent increases in cellular cAMP levels with the expected growth arrest and cytolysis . Involvement of cAMP-dependent protein kinase in this type of desensitization was suggested by the observation that S49 kincells were not desensitized by long term incubation with 3 nM epinephrine . Further, low concentrations of dibutyryl cAMP mimicked the effect of low level epinephrine treatment . We conclude that circulating levels of epinephrine in intact animals are sufficiently high to cause desensitization in cells with sensitivities to the catecholamines in the same range as that of the S49 lymphoma cell in vitro . We would predict that cells with those characteristics would always be at least partially desensitized in vivo.

J Cell Biol, 1989 Sep, 109(3), 1037 - 46
Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor; Einstein R et al.; Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes . The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography . 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated . In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated . The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment . Transition from the high to low density state was rapid and was not inhibited by cycloheximide . Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state . Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated . The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.

Calcif Tissue Int, 1989 Sep, 45(3), 193 - 7
Effect of L-lysine on cytosolic calcium homeostasis in cultured human normal fibroblasts; Civitelli R et al.; L-lysine, a cationic essential amino acid, has been reported to affect calcium transport in both intestine and kidney . In order to investigate whether this effect is associated with changes in cytosolic calcium homeostasis, we studied the effect of L-lysine deprivation on intracellular calcium concentration ({Ca2+}i), as well as 45Ca efflux and accumulation in normal human fibroblasts . Steady state {Ca2+}i, measured using fura-2 fluorescence in cells cultured for 18 hours in a L-lysine-free medium, was significantly higher than in cells grown in the presence of as little as 4 microM L-lysine . L-lysine deprivation also led to a significant decrease of 45Ca fractional efflux compared with cells grown in complete medium . This effect was paralleled by a significant decrease in 45Ca accumulation . Lack of L-arginine from the growth medium for the same time period had no effect on either {Ca2+}i, 45Ca efflux, or 45Ca accumulation rate . Presumably, the lack of L-lysine for a significant amount of time impairs the active mechanisms of calcium extrusion, which is only partially compensated by a reduction of calcium accumulation rate . This leads to an increased steady state {Ca2+}i . It is concluded that L-lysine is an important modulator of cytosolic calcium homeostasis.

J Bacteriol, 1989 Sep, 171(9), 4923 - 9
Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads; Rosenbluh A et al.; Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer . In growth medium, the cells multiplied, glided to the periphery, and then filled the beads . In sporulation buffer, up to 90% of the cells lysed and ca . 50% of the surviving cells formed resistant spores . A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent . Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed . A minimum of ca . 4 cells per microbead was necessary for efficient lysis and sporulation to proceed . Increasing this number accelerated the lysis and sporulation process . No lysis occurred when an average of 0.4 cell was entrapped per bead . Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells . Sporulation and lysis also depended upon the cell density in the culture as a whole . The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M . xanthus.

Immunol Lett, 1989 Aug, 22(2), 167 - 71
Growth of B cell colonies independent of T cell contact; Fernandez LA et al.; One of the methods of obtaining B cell colonies involves physically admixing B cells in growth medium containing mitogens with accessory T cells treated with mitomycin C or low dose irradiation to prevent T cell colony formation . However, under these conditions T cells still have the capacity to form colonies in methylcellulose, which is most frequently used for colony assays, and B cells recovered from the colonies are contaminated by the large number of added T cells . Therefore, we have developed a method of growing B cell colonies by physically separating the enriched B cells from the T cells by using millicell-HA inserts (Millipore) . The requirement for mitogens, culture conditions, development, and number of B cell colonies formed were similar to those observed with admixture of T and B cells . The advantage of this system is that it permits growth of B cell colonies without the confounding influence of T cell proliferation or treatment of T cells with mitomycin or irradiation, and relatively pure B cell colonies can be recovered for further characterization or functional studies.

Photochem Photobiol, 1989 Aug, 50(2), 185 - 91
Photoreactivation of lethal damage and damage leading to chromatid deletions induced in G1 phase hamster x Xenopus hybrid cells by UV; Kulp SQ et al.; A86 Xenopus cells, cloned from a Xenopus line that exhibited a high level of photoreactivation of UV-induced lethal damage, and V79M1 hamster cells, cloned from a hamster line that did not exhibit efficient photoreactivation of such damage, were fused to produce the V79M1 x A86 cell line--a hybrid line in which approximately 84% of the cells contained the entire V79M1 and A86 genomes . Ultraviolet and UV plus photoreactivation fluence-survival relations were then determined and compared for hybrid and parental G1 phase cells in a first attempt to elucidate interactions of the parental genetic potentials for photoreactivation in the hybrid . Specifically, it was anticipated that the combined V79M1 and A86 genomes in the hybrid would produce photoreactivating enzymes sufficient to efficiently photoreactivate UV-induced lethal damage in both A86 and V79M1 DNA and little difference would be observed in the levels of photoreactivation exhibited by V79M1 x A86 and A86 G1 phase cells . To the contrary, the level of photoreactivation observed for the hybrid did not closely approach that observed for the A86 line . To assist in the interpretation of this somewhat unexpected observation, three additional studies were performed: (1) comparison of 'optimal' schemes for photoreactivation of UV-induced lethal damage in the hybrid and parental G1 phase cells, (2) comparison of the effects of some different types of growth medium on photoreactivation of UV-induced lethal damage in hybrid and parental G1 phase cells, and (3) comparison of the levels of photoreactivation of UV-induced chromatid deletions in the V79M1 and A86 chromosomes of G1 phase hybrid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biol, 1989 Aug, 109(2), 789 - 98
Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms; Doherty P et al.; Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells . Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons . The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons . Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation . 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation . These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.

Radiat Res, 1989 Aug, 119(2), 338 - 47
Cell cycle progression delay in conditioned medium does not play a role in the repair of X-ray damage in Chinese hamster V79 cells; Reddy NM et al.; We tested our hypothesis that the lower survival of X-irradiated cells in growth medium (GM) relative to that in conditioned medium (CM) is due to differences in nutrient concentration levels rather than to differential effects on cell progression and growth . Chinese hamster V79 cells in log and unfed plateau phase, grown in Eagle's minimal essential medium (MEM) with 15% serum (100% GM), were irradiated . Before plating, cells were incubated in situ in various concentrations of MEM with serum (GM, normal cell progression) or MEM without serum or in CM (no cell progression) . Cell survival was the lowest in 100% MEM with or without serum and increased with the decrease in MEM and serum concentrations, reaching a plateau in 40% MEM or 40% growth medium (40% MEM with 6% serum), similar to that in conditioned medium . Growth kinetics was the same in 40 and 100% growth medium, but the D0 of cells in 40% growth medium was higher than that of cells in 100% GM . Similarly, the D0 of cells in 40% MEM was higher than that of cells in 100% MEM, although cell progression was absent in both media . The radiation sensitivity of cells was the same in 40% GM with progression and in 40% MEM and CM with no progression . Cells in low-nutrient media were flatter than those in 100% MEM or GM . There was a correlation between the nutrient concentration in the medium postirradiation and the D0 . This correlation was independent of the presence or absence of serum and thus independent of cell cycle progression . The cell morphology which is dependent on the nutrient concentration appears to influence the ability of a fraction of cells to repair their radiation damage.

Blood, 1989 Aug 1, 74(2), 602 - 8
Release of soluble transferrin receptor from the surface of human leukemic HL60 cells; Chitambar CR et al.; Information regarding transferrin (Tf) receptor degradation is largely incomplete . HL60 cells were shown to release to their growth medium a Tf-binding protein which could be immunoprecipitated by anti-Tf receptor monoclonal antibodies (MoAbs) B3/25 and OKT9 . Soluble Tf receptor was detected in the medium within one hour of replating of cells, and its release was inhibited at 4 degrees C . The affinity of Tf for the soluble receptor released by cells (kd = 2.3 x 10(-10) mol/L) was slightly lower than its affinity for the detergent-solubilized cellular receptor (kd = 1.2 x 10(-10) mol/L) . 125I-Tf internalized and released by cells subsequently bound to Tf receptor released by the same cells, and soluble Tf receptor in the conditioned medium (CM) inhibited 125I-Tf binding to intact cells . The soluble Tf receptor isolated from the CM was smaller (78,000 daltons) than the cell surface receptor (94,000 daltons) when analyzed by gel electrophoresis under reducing conditions . Isolated cell membranes readily released soluble receptor; however, this release could be blocked by protease inhibitors . The soluble Tf receptor may represent the extracytoplasmic domain of the cellular Tf receptor released from the surface of HL60 cells through proteolytic cleavage by a membrane-based protease.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6018 - 22
Induction of an antioxidant protein of Saccharomyces cerevisiae by O2, Fe3+, or 2-mercaptoethanol; Kim IH et al.; A soluble 27-kDa protein from Saccharomyces cerevisiae specifically prevents the inactivation of various enzymes caused by a nonenzymatic Fe3+/O2/thiol mixed-function oxidation system but not by mixed-function oxidation systems in which the thiol component is replaced by another electron donor-e.g., ascorbate . In this report, using a 125I-labeled monospecific antibody against the 27-kDa protein, we measured changes in the 27-kDa protector protein in response to changes in oxidative stress and heat shock . With a shift from an anaerobic (95% N2/5% CO2) to a hyperaerobic (95% O2/5% CO2) atmosphere, a 3-fold increase was observed . This increase was prevented by cycloheximide, indicating that the induction requires new protein synthesis . The antioxidant protein synthesis was also significantly enhanced by the addition of either 2-mercaptoethanol or Fe3+ to the growth medium . Radioimmunoassay results also show that the antioxidant protein is an abundant protein, as it constitutes 0.7% of total soluble protein from yeast grown aerobically . Immunoblotting experiments revealed that rat tissues also contain a 27-kDa protein that can be specifically recognized by antibodies against the yeast protein . These results suggest that in vivo induction in yeast of the 27-kDa protein may represent an adaptive response that evolved to protect cells against damage caused by thiol-dependent mixed-function oxidation systems, and the antioxidant protein is conserved in mammalian tissues . A heat shock applied to yeast did not cause any significant increases in the concentration of the 27-kDa protein.

Mol Cell Biol, 1989 Aug, 9(8), 3292 - 8
Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene; Thomas D et al.; In Saccharomyces cerevisiae, the MET25 gene encodes O-acetylhomoserine sulfhydrylase . Synthesis of this enzyme is repressed by the presence of S-adenosylmethionine (AdoMet) in the growth medium . We identified cis elements required for MET25 expression by analyzing small deletions in the MET25 promoter region . The results revealed a regulatory region, acting as an upstream activation site, that activated transcription of MET25 in the absence of methionine or AdoMet . We found that, for the most part, repression of MET25 expression was due to a lack of activation at this site, reinforced by an independent repression mechanism . The activation region contained a repeated dyad sequence that is also found in the promoter regions of other unlinked but coordinately regulated genes (MET3, MET2, and SAM2) . We show that the presence of the two dyads is necessary for maximal gene expression . Moreover, we demonstrate that in addition to this transcriptional regulation, a posttranscriptional regulation, probably targeted at the 5' region of mRNA, is involved in MET25 expression.

EMBO J, 1989 Aug, 8(8), 2203 - 7
Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells; Montarras D et al.; We have examined the expression of MyoD1, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts . These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation . Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation . We show that while expression of MyoD1 is constitutive in permissive cells, no trace of MyoD1 transcripts is found in inducible cells at the myoblast stage . In these cells, however, expression of MyoD1 accompanies differentiation . This indicates that MyoD1 may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells . Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation.

Health Phys, 1989 Aug, 57(2), 309 - 14
The uptake of TcO-4 by plants: a mathematical description; Van Loon LR et al.; A model describing the uptake of TcO-4 by spinach plants was developed . The equation relates both plant and soil parameters (e.g., growth, metabolism, concentration of TcO-4 and composition of the growth medium) to the concentration of Tc in the shoot of the plant . As the soil solution is the medium from which plants obtain nutrients and non-nutrients, the modeling parameters have been obtained from uptake experiments using nutrient solutions (= simulated soil solutions) as the growth medium . Two important model assumptions are: 1) that an equilibrium exists between TcO-4 in the plant and the growth medium and 2) that the leaf TcO-4 metabolism is a pseudofirst order reaction occurring in a non-constant volume.

Health Phys, 1989 Aug, 57(2), 239 - 45
Bioaccumulation and chemical modification of Tc by soil bacteria; Henrot J; Bioaccumulation and chemical modification of pertechnetate (TcO4-) by aerobically and anaerobically grown soil bacteria and by pure cultures of sulfate-reducing bacteria (Desulfovibrio sp.) were studied to gain insight on the possible mechanisms by which bacteria can affect the solubility of Tc in soil . Aerobically grown bacteria had no apparent effect on TcO4-; they did not accumulate Tc nor modify its chemical form . Anaerobically grown bacteria exhibited high bioaccumulation and reduced TcO4-, enabling its association with organics of the growth medium . Reduction was a metabolic process and not merely the result of reducing conditions in the growth medium . Association of Tc with bacterial polysaccharides was observed only in cultures of anaerobic bacteria . Sulfate-reducing bacteria efficiently removed Tc from solution and promoted its association with organics . Up to 70% of the total Tc in the growth medium was bioaccumulated and/or precipitated . The remaining Tc in soluble form was entirely associated with organics . Pertechnetate was not reduced by the same mechanism as dissimilatory sulfate reduction, but rather by some reducing agent released in the growth medium . A calculation of the amount of Tc that could be associated with the bacterial biomass present in soil demonstrates that high concentration ratios in cultures do not necessarily imply that bioaccumulation is an important mechanism for long-term retention of Tc in soil.

J Bacteriol, 1989 Aug, 171(8), 4385 - 94
Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose; Moore MD et al.; We have constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides . We introduced TnphoA into the genome of R . sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6) . Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants . Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate . The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined . The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R . sphaeroides genomic DNA capable of complementing individual mutants have also been isolated . The usefulness of this approach in studying gene activity in R . sphaeroides is discussed.

Neurosci Lett, 1989 Jul 31, 102(2-3), 268 - 72
In vitro kinetics of a newborn rat astroglia-derived neuronotoxic activity; Leprince P et al.; A low-molecular weight astrocyte-derived neuronotoxic activity (ANTA) was detected, using a colorimetric bioassay of cell survival, by its effect on cultured granule cells . This neuronotoxic activity was found to be released rapidly from newborn rat astrocytes in culture upon incubation in 50 mM K+-containing growth medium . The release by astrocytes could be induced repetitively by successive incubations in high-K+ medium alternating with incubations in normal medium . Astrocytes were also found to inactivate rapidly isobutanol-extracted ANTA in normal K+-containing growth medium . Kinetic studies showed that ANTA induces a slow (greater than 12 h) degeneration of cultured granule cells . ANTA is shown here to be an intermediate of normal astrocyte metabolism and to display appropriate kinetic characteristics compatible with its proposed role in inducing part of the delayed neuronal loss that occurs after a brain injury (secondary neuronal death).

J Immunol Methods, 1989 Jul 26, 121(2), 167 - 74
Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells; Meilhoc E et al.; The kinetics of antibody synthesis and secretion from a murine hybridoma cell line were studied using measurements of total cell-associated IgG, surface IgG, and IgG secreted into the medium . Kinetic analysis of IgG secretion demonstrates approximately constant secretion rate per viable cell over the entire batch cultivation . A correlation was observed (r2 = 0.74) between mean surface immunofluorescence and the total cell-associated IgG determined by ELISA of detergent-extracted cell lysates . No correlation was found between specific secretion rate and mean surface IgG level estimated by immunofluorescence flow cytometry measurements . Material balances on cellular IgG demonstrated that about 7% of the antibody which was synthesized during exponential batch growth was not released to the growth medium . Distributions of single-cell surface antibody content showed two subpopulations, one with very low surface IgG . The fraction of the population with low surface IgG increased throughout a batch cultivation.

Biochem J, 1989 Jul 15, 261(2), 649 - 53
Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences; Gaston K et al.; Binding of the Escherichia coli CRP protein to DNA fragments carrying nucleotide sequences closely corresponding to the consensus is very tight with a dissociation time of over 2 h in our conditions . The concentration of cyclic AMP required for this binding is below the physiological range of intracellular cyclic AMP concentrations . Changes in nucleotide sequence at positions that are not well-conserved between different naturally-occurring CRP sites allow a more rapid dissociation of CRP-DNA complexes . There is an inverse correlation between the stability of CRP binding to sites in vitro and the repression by glucose of expression dependent on these sites in vivo: expression that is dependent on the tighter binding sites cannot be repressed by the inclusion of glucose in the growth medium.

Tsitologiia, 1989 Jul, 31(7), 818 - 23
{Characteristics of action of precursors of carcinogenic nitroso-compounds on cell cultures}; Osin'kovskaia ND et al.; On the primary rat lung cell cultures, a study was made of the transforming action of sodium nitrite (NN) and amidopyrine combination with the control for formation of carcinogen--N-nitrosodimethylamine (NDMA) in growth medium . The manifestation of transformation was registered by appearance of morphological changes and multilayer growth foci . As criteria for evaluation after 48 hour treatments with NDMA, NN and AP, were used DNA-synthetizing activity of cells, mitotic index, frequency of mitoses pathology, monolayer density . The effects of transforming dose of NN alone and in combination with AP were the same . But malignization (tumor development in newborn rats in the points of cell suspension inoculation) took place only after administration of NN in combination with AP, when carcinogen was formed . Theophylline decreased the action of NN-AP combination.

Pflugers Arch, 1989 Jul, 414(3), 265 - 72
Voltage- and time-dependent chloride currents in chick skeletal muscle cells grown in tissue culture; Steele JA; Membrane chloride currents in chick skeletal muscle cells grown in tissue culture were studied by use of the whole cell variation of the patch electrode voltage clamp technique . Small diameter myoballs were obtained by adding colchicine to the growth media . To isolate the currents through the chloride channels, the currents through the sodium, calcium and potassium channels were minimized . With symmetrical chloride concentrations bathing the membrane, inward currents were activated by depolarizations above -45 mV . Above 0 mV, the currents became outward . The reversal potential for the currents shifted with the chloride concentration gradient in a manner consistent with the Nernst relation, indicating that the currents were predominantly carried by chloride ions . The instantaneous current-voltage relation obtained from tail current data was linear . The relationship between conductance and membrane potential was sigmoid . The conductance activated above -45 mV, increased steeply between -45 and -10 mV and saturated above +20 mV . Over the range of potentials where the conductance was just beginning to activate, the conductance increased e-fold for a 7 mV depolarization . The currents activated with an exponential time course and did not decline during step depolarizations . Tail currents declined slowly as the sum of two exponential components . The currents were reversibly suppressed by 100 microM SITS and were irreversibly suppressed by 10 microM DIDS.

J Parenter Sci Technol, 1989 Jul-Aug, 43(4), 183 - 7
The effect of refrigeration and mixing on detection of endotoxin in parenteral drugs using the Limulus Amebocyte Lysate (LAL) test; Guilfoyle DE et al.; Prior to testing for the presence of bacterial endotoxin, parenteral products are handled and stored in a variety of ways . Two incidents, detected by the U.S . Food and Drug Administration, revealed that differences in product handling and storage may have played a role in causing analytical discrepancies in the testing of identical samples . The testing procedure was the USP Bacterial Endotoxin test using Limulus Amebocyte Lysate (LAL) reagent . Consequently, an evaluation was made at the two principal factors that contributed to the suspected analytical anomaly . The factors were sample storage and the degree of agitation prior to sample analysis . Additional variables such as bacterial growth medium and adsorption potential of endotoxin by rubber stoppers were also evaluated . It was found that neither the medium employed to grow the E . coli endotoxin nor the storage temperature of the spiked solutions were problematic . However, it was shown that 20-40% of the spiked endotoxin was lost due to non-agitation of solution in vials in which the solution was in contact with the rubber stoppers . A suggested remedy for this problem is to store intact product containers in an upright position and to establish a uniform mixing procedure prior to endotoxin assay.

Radiat Res, 1989 Jul, 119(1), 88 - 100
Interaction between radiation and drug damage in mammalian cells . IV . Radiation response of adriamycin-resistant V79 cells; Belli JA; Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties . A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve . Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers . A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury . LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties . These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics . LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium . Reverted cells also lost their large capacity for sublethal damage . It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.

Cell Tissue Res, 1989 Jul, 257(1), 129 - 36
Effect of progesterone on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium; Chaminadas G et al.; The pattern of proteins synthesized and secreted in response to progesterone by guinea-pig endometrial epithelial cell cultured with estradiol-17 beta was investigated . Glandular epithelial cells were maintained in culture for 3 days on growth medium, then washed three times with a steroid-free medium . After this period, 2 x 10(-8) M estradiol-17 beta or 2 x 10(-8) M estradiol-17 beta plus 5 x 10(-7) M progesterone were added to the medium for 48 h . To study biochemical changes, the proteins in medium and in cells were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography . The addition of progesterone to estradiol-17 beta in the culture medium caused a change in the patterns of cellular and secreted proteins: one-dimensional gel electrophoresis showed that variation of 8 cellular proteins and 12 secreted proteins was caused by progesterone . Induction of individual proteins by progesterone treatment was observed by two-dimensional gel electrophoresis: one cellular protein (Mr 49,000; pI 5.90) and one secreted protein (Mr 14,300; pI 4.80) were specifically induced and might serve as markers of progesterone action.

J Bacteriol, 1989 Jul, 171(7), 4031 - 7
Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+; Huyer M et al.; Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm . The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays . Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells . The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity . The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A . vinelandii . The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000 . The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics . The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH . These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A . vinelandii.

In Vitro Cell Dev Biol, 1989 Jul, 25(7), 592 - 600
Retention of differentiated characteristics in human fetal keratinocytes in vitro; Haake AR et al.; Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin . This is due in part to difficulties in culturing human fetal epidermal keratinocytes . We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates . To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation . Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins . However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane . More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts, including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins . The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis.

Biochem Biophys Res Commun, 1989 Jun 30, 161(3), 1312 - 8
Human tumor cells resistant to verapamil; Huber KR et al.; The efficacy of the calcium channel blocker verapamil for enhancing at low concentrations the cytotoxicity of unrelated antineoplastic drugs and for inhibiting at high concentrations cell proliferation has stimulated interest in the underlying mechanisms of these two diverse effects . We have selected two human brain tumor cell lines (a TE671 medulloblastoma and a A172 glioma line) for resistance against 100 uM verapamil to aid in the elucidation of the mechanism of verapamil's antiproliferative effect . Our first experiments on the selected TE671 medulloblastoma cells show that, in the presence of 100 uM verapamil, these cells grow at a rate similar to that observed for the sensitive cells in the absence of verapamil . This resistant clone continues to exhibit resistance toward verapamil for at least three days after the verapamil has been removed from the growth medium . In contrast to the sensitive cells, the resistant cells show only slight cell cycle phase alterations after removal of verapamil from the growth medium . This, together with an unchanged c-myc gene expression after removal of verapamil, indicates a stable phenotypic alteration that is responsible for the exhibited resistance toward the antiproliferative effects of the drug . Experiments designed to elucidate the mechanism of resistance showed that these cells are not cross-resistant to the antineoplastic drugs vincristine and adriamycin . Also, the resistance is not accompanied by increased amounts of the 170-180 kDa P-glycoprotein that has been implicated in resistance phenomena of cancer cells towards antineoplastic drugs.

Eur J Biochem, 1989 Jun 15, 182(2), 213 - 21
Functional analysis of the signal-sequence processing site of yeast acid phosphatase; Monod M et al.; A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented . The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement . Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e . alanine, glycine, cysteine, serine or threonine . Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell . Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence . The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.

J In Vitro Fert Embryo Transf, 1989 Jun, 6(3), 164 - 7
Bovine serum albumin (BSA) can replace patient serum as a protein source in an in vitro fertilization (IVF) program; Benadiva CA et al.; Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures . During an 11-month period 127 patients treated for in vitro fertilization had either their serum (N = 71) or bovine serum albumin (BSA; N = 56) used as the protein source in the insemination and growth media . Ham's F-10 + 0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media . Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively . Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups . Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA . Likewise, rates of abnormal fertilization, cleavage, and pregnancy were similar in both groups . In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%) . Cleavage rates for the two groups were similar and the percentage of embryos developed to greater than or equal to 4 cells did not differ significantly . We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.

J Pathol, 1989 Jun, 158(2), 123 - 9
Cell death by apoptosis in acute leukaemia; Baxter GD et al.; We have previously demonstrated that when freshly isolated childhood T-cell acute lymphoblastic leukaemia cells are incubated in growth medium after isolation from blood, chromatin is rapidly cleaved into nucleosomal sized fragments that are multiples of 200 bp . The fragmentation is similar to that observed in other types of cells undergoing apoptosis or programmed cell death . In this study we describe a more comprehensive approach to the study of DNA fragmentation in leukaemia . Fragmentation was observed in freshly isolated cells from patients with T-cell acute lymphoblastic leukaemia and in one with common acute lymphoblastic leukaemia . Frozen samples of T-cell acute lymphoblastic leukaemia, common acute lymphoblastic leukaemia, and acute myeloid leukaemia cells also showed fragmentation of DNA . However, no fragmentation was evident in normal leukocytes treated under the same conditions . Ultrastructural studies on the isolated leukaemia cells demonstrate that the chromatin cleavage observed biochemically is associated with morphological changes characteristic of apoptosis.

Brain Res Dev Brain Res, 1989 Jun 1, 47(2), 171 - 9
Levels of nerve growth factor secreted by rat primary fibroblasts and iris transplants are influenced by serum and glucocorticoids; Houlgatte R et al.; Previous work performed with mouse fibroblast-like L cells has shown that the level of expression of NGF gene is modulated in these transformed cells by the composition of the growth medium . Glucocorticoids were found to exert a down-regulation on NGF production, while serum stimulated the synthesis of the factor . The contrasting effects of serum and dexamethasone were further investigated in cultures of primary rat fibroblasts or in iris transplants . ELISA assays of NGF released by fibroblasts or by transplanted irides showed that both experimental systems responded to dexamethasone by a 4-5-fold decrease of the amounts of secreted factor . Half-maximal effect took place at a concentration of 3-5 X 10(-9) M, a value close to the dissociation constant of the glucocorticoid receptor in fibroblasts . The glucocorticoid did not influence the secretion of macromolecules . Assays of NGF mRNA performed at a concentration of 10(-7) M dexamethasone indicated that the steroid decreased the pool of NGF transcripts in either experimental systems . In contrast to dexamethasone, serum induced a 4-fold enhancement of the amounts of factor secreted by fibroblasts . This effect was reproduced with serum that was previously heat-treated at mild acidic pH, or with a macromolecular fraction of this heat-treated serum which contains an effector promoting NGF synthesis in L cells . The fact that promotion of NGF synthesis takes place in primary cells raises the possibility that this process may also occur in vivo, for instance following disruption of vasculature, as a part of a wound mechanism . Data collected with iris transplants provide some support to this interpretation.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1989 Jun, 171(6), 3406 - 11
Identification of cyclic intermediates in Azorhizobium caulinodans nicotinate catabolism; Kitts CL et al.; In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source . Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source . Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth . Therefore, A . caulinodans wild type is NAD+ auxotrophic . As the first two intermediates in A . caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified . These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra . The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2 . From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source . A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport . These catabolic reactions have not previously been witnessed together in the same organism . A . caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.

Infect Immun, 1989 Jun, 57(6), 1862 - 4
Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium; Catrenich CE et al.; The phenotypic difference between virulent and avirulent Legionella pneumophila with regard to growth on supplemented Mueller-Hinton (SMH) agar was investigated . Defined populations of virulent and avirulent L . pneumophila were inoculated onto hybrid growth media containing the combination of SMH agar components with potassium phosphate-buffered charcoal-yeast extract agar . The casein acid hydrolysate component of SMH agar was found to be inhibitory to the growth of virulent but not avirulent cells . The selectively inhibitory component of the casein acid hydrolysate was identified as NaCl.

Anat Rec, 1989 Jun, 224(2), 234 - 41
Mineral phases of calcium phosphate; Nancollas GH et al.; Many studies of calcium phosphate precipitation have been made using relaxation techniques in which the concentrations of the lattice ions are allowed to decrease as equilibrium is approached . Since the nature of the phases that form depend markedly on the solution composition, this decrease can lead to concomitant phase transformations during the crystallization experiments . The results of the present constant composition (CC) studies show that defect apatites may be formed under conditions of sustained supersaturation with a non-stoichiometric coefficient dependent on the pH of the growth medium . An important factor in analyzing these experiments is the initial surface modification and ion-exchange processes involving H+ and Ca2+ ions after inoculation of the supersaturated solutions . Thereafter, active growth sites may be eliminated as the crystals undergo lattice perfection . Transformation of dicalcium phosphate dihydrate to octacalcium phosphate, involving dissolution and subsequent nucleation and growth of the new phase, is also influenced by surface roughening of the initial phase . Typical inhibitors that reduce the rate of growth of seed crystals in supersaturated solutions may actually induce the nucleation of calcium phosphate phases when immobilized on inert surfaces . This may be a factor in the modulation of crystal growth in many biological systems.

Neurochem Res, 1989 Jun, 14(6), 547 - 54
Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: inhibition by calcium ion and dependence upon membrane protein; Leskawa KC et al.; Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined . Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10(-4) M {3H}GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin . Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution . When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca2+ was lost and total incorporation into membranes was lowered by approximately one order of magnitude . Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes . When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with {3H}leucine . The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins . These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.

J Bacteriol, 1989 Jun, 171(6), 2949 - 55
DNA-binding properties of the transcription activator (OmpR) for the upstream sequences of ompF in Escherichia coli are altered by envZ mutations and medium osmolarity; Forst SA et al.; Expression in Escherichia coli of the genes that encode the major outer membrane porin proteins (OmpF and OmpC) is regulated by the transcription activator protein OmpR and the receptorlike protein EnvZ, which is located in the inner membrane . Using synthesized oligonucleotide fragments containing the OmpR-binding site of ompF, we show that soluble extracts and partially purified OmpR derived from both the parent strain grown in nutrient broth plus 20% sucrose and the envZ11 strain grown in nutrient broth produced high-affinity DNA-binding activity, whereas soluble extracts from the parent strain grown in nutrient broth produced low-affinity binding . We also show that the soluble extracts from the envZ22(Am) strain grown in nutrient broth did not produce detectable bound forms of the ompF fragments, but low levels of DNA binding were detected with soluble extracts of the envZ22 strain grown in nutrient broth plus sucrose . In addition, the time course of the repression of OmpF synthesis produced by a shift to high-osmolarity growth medium was correlated with an increase in the DNA-binding affinity of soluble extracts to the ompF fragment . These results provide evidence that envZ function influences the DNA-binding activity of OmpR and suggest that high-affinity binding of OmpR to the upstream sequences of ompF is correlated with the repression of OmpF production.

Kansenshogaku Zasshi, 1989 Jun, 63(6), 575 - 83
{An experimental consideration of the improved (1974) Kelsy-Sykes test for bactericidal effect of disinfectants}; Akiyama S et al.; In 1974, Kelsey and Maurer proposed the improved Kelsey-Sykes test as an evaluation method for bactericidal effect of all kinds of disinfectant . We examined the necessity of culturing test organism with the fixed special broth, preparing organism population and using the special container called the universal container proposed in this test . We compared these test results with the phenol coefficient method used usually as an evaluation method for bactericidal effect of disinfectants, and investigated its availability and experimental procedure: the results are as follows . 1) There was not difference between growth medium according to K-S test (Bacto Synthetic Broth A.O.A.C . Code No . 0352) and our nutrient broth (Nissui Nutrient Broth Lot No . 065210) in growth support for the test organisms . 2) There was not difference in test results of bactericidal effect between the standard hard water and whole broth culture used as test organisms suspension or organism suspension with the pellicle of P . aeruginosa and without one . 3) The result using the usual operating test tube was the same as when the universal container was used . 4) The procedure of K-S test was complex in comparison with the PC test method, but was able to presume the usual use-dilution . The K-S test had an advantage in this point.

Eur J Cell Biol, 1989 Jun, 49(1), 92 - 8
A growth hormone-vesicular stomatitis virus G hybrid protein is rapidly degraded in lysosomes following transport to the cell surface; Rizzolo LJ; We have expressed the hybrid protein, GHG3, in baby hamster kidney cells to study protein turnover . GHG3 contains the cytoplasmic and transmembrane domains of vesicular stomatitis virus G protein linked to the C-terminus rat growth hormone . Turnover of GHG3 was prevented by lysosomal inhibitors (leupeptin, chloroquine, primaquine or monensin), while the accumulated GHG3 was localized to intracellular vesicles, results indicating that degradation occurred in lysosomes . The kinetics of degradation at 34 degrees C were determined in pulse-chase studies of metabolically labeled cells . After a lag period of 1 h, degradation was rapid (t1/2 = 1.25 h) . The fate of GHG3 during the lag period was determined by immunofluorescence . We detected GHG3 on the cell surface when growth hormone antiserum was added to the growth medium 90 min prior to fixation and staining . No staining was observed if protein synthesis was inhibited with cycloheximide 90 min prior to the addition of growth hormone antiserum, a result indicating that GHG3 was rapidly removed from the cell surface . Unless the cells were pretreated with cycloheximide, antiserum was also detected in intracellular vesicles, which showed that GHG3 was endocytosed . These data indicate that a pool of GHG3 is transported rapidly to the cell surface, endocytosed and with little or no recycling directed to lysosomes for degradation.

Hybridoma, 1989 Jun, 8(3), 331 - 6
A live cell enzyme-linked immunosorbent assay for detecting human hepatoma membrane antigens; Gaffar SA et al.; Live cell enzyme-linked immunosorbent (ELISA) and fixed cell indirect immunofluorescence (IF) assays were compared to screen mouse hybridomas producing immunoreactive monoclonal antibodies against cell membrane antigens expressed on Ha22T, a human hepatoma cell line . While performing live cell ELISA, two parameters were tested to improve the viability of the target cells . The first parameter was the inclusion of growth medium in the assay buffers, and the second was performing the assay incubations at 37 degrees C in an incubator containing 5% CO2 in the air . Fixed cell IF detected and classified 46% of the hybridomas secreting monoclonal antibodies reactive with membrane, cytoplasm, cytoskeleton, and nuclear antigens of Ha22T cells . Fixed cell IF was able to reveal mixtures of two or more hybridomas growing in the same well secreting antibodies to different cell organelles . The live cell ELISA, on the other hand, identified 12 additional membrane reactive monoclonal antibodies from the hybridoma supernatants that were not reactive by IF . These results disclose that cell fixation procedures used for IF either completely or partially inactivated some of the cell membrane antigens . We, therefore, propose the use of a combination of immunoassays to select the maximum number of hybridomas secreting useful monoclonal antibodies from somatic cell fusions.

J Bacteriol, 1989 Jun, 171(6), 3511 - 7
Mapping of the Escherichia coli acid glucose-1-phosphatase gene agp and analysis of its expression in vivo by use of an agp-phoA protein fusion; Pradel E et al.; The agp gene of Escherichia coli encodes an acid glucose-1-phosphatase, one of the numerous phosphatases optimally active between pH 4 and 6 found in the periplasmic space of this bacterium . An agp-phoA protein fusion linked to a gene conferring kanamycin resistance was inserted into the chromosome in place of agp by homologous recombination and was mapped to minute 22.6 . Because the activity of glucose-1-phosphatase cannot be measured accurately in whole cells, the alkaline phosphatase activity of the agp-phoA hybrid protein was used to monitor the expression of the chromosomal agp gene . The expression of agp was subject to catabolite repression but was unaffected by the concentration of inorganic phosphate in the growth medium . The product of the agp gene was required for growth on glucose-1-phosphate as the sole carbon source, a function for which alkaline phosphatase or other acid phosphatases cannot substitute.

J Autoimmun, 1989 Jun, 2(3), 269 - 82
Investigations of intrinsic abnormalities in DNA-specific B lymphocytes from autoimmune mice; Aldo-Benson M; In murine models of systemic lupus erythematosus and in many humans with SLE, antibodies against native DNA (dsDNA) are a major contributor to the pathogenesis of the disease . Loss of self-tolerance to the DNA antigen may be associated with B-cell defects or regulatory cell dysfunction . We have developed B-cell lines with specificity for the antigen DNA, from both the autoimmune BWF1 mouse strain and from the non-autoimmune BALB/c strain, to use in the investigation of inherent B-cell defects in autoimmunity . Six BWF1 cell lines and five BALB/c cell lines which are free of Thy1.2+ cells and esterase positive cells, and have between 35 and 89% rosetting with dsDNA-SRBC targets, have been propagated in vitro for 24-36 months . The cells are non-malignant, growth-factor dependent and have no antigen or mitogen in the growth medium . Lyt-1 positive cells are found in the cell lines, but Lyt-1 negative cells are also present . They respond to the antigen DNA-HRBC when EL-4 supernatant is present in culture, and the peak of the plaque-forming cell (PFC) response is the same for both strains . When cells from both strains are cultured with varying amounts of T-cell factors, there is no difference in spontaneous antibody-forming cell (AFC) formation or in response to anti-mu stimulation between BWF1 and BALB/c strains . BALB/c spleen cells do not respond to DNA-HRBC in this culture system, but BWF1 spleen cells, as well as cell line cells from both strains, respond to this antigen . T cells from non-responding BALB/c spleen and responding BWF1 spleen are able to suppress the immune response to DNA-HRBC of cell line B cells from both strains . Propagating B-cell lines in the presence of DNA for 2 weeks stimulates BWF1 cell line cells, but suppresses the response of BALB/c cell lines to antigen.

Arch Biochem Biophys, 1989 Jun, 271(2), 315 - 22
Uptake, intracellular binding, and excretion of polyamines during growth of Neurospora crassa; Davis RH et al.; In Neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight . We wished to know what determines these pool sizes . In the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system . In a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth . However, this was true only after an intracellular "sink," with a capacity equal to the amount of spermidine found in wild-type cells, had been saturated . We speculate that internal anionic binding sites, detectable in permeabilized cells, sequester virtually all of the spermidine normally found in exponentially growing N . crassa . Further evidence for this view was that in mature, stationary cultures, excess spermidine is excreted . Putrescine is also excreted if its concentration in the cell is abnormally high . The control of pool size by intracellular binding and excretion may be an advantage in this pathway, because feedback inhibition does not prevail, enzyme regulation is by comparison slow, and excessive polyamines are toxic.

J Cell Biol, 1989 Jun, 108(6), 2107 - 15
Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells; Kumar RV et al.; Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium . This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers . To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77) . BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM . This effect was reversed by calcium but not by other cations tested . The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes . Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3 . Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.

Cancer Res, 1989 May 15, 49(10), 2679 - 82
Stimulation of growth in human and murine cells by adriamycin; Vichi P et al.; Adriamycin causes a variety of biological actions and is an effective cytotoxic agent against proliferating cells . In this paper we show that the drug is not limited in its action solely to cytotoxicity, but can also stimulate cell growth under the appropriate conditions . Using the survival assay of cloning in soft agar, we present data showing that the conditions for Adriamycin-induced growth stimulation are that the drug be in a subtoxic concentration range of 10(-10)-10(-9) M (greater than 10(-8) M causes cytotoxicity) and that the growth medium be suboptimal . This latter condition is satisfied by either growing cells for an extended period in order to exhaust the growth supporting capacity of the medium, or by growing the cells at low (less than 10%) serum concentrations . Several active anthracycline congeners also have the ability to stimulate growth . The results indicate that the cytotoxic anticancer agent Adriamycin can stimulate the proliferation of some cells.

Biochim Biophys Acta, 1989 May 10, 1011(2-3), 102 - 9
Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors; Bigler RD et al.; A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI) . Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI . Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability . While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a . A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion . Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly . In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density . High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol . This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth . These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.

Neurosci Lett, 1989 May 8, 99(3), 263 - 7
Biological actions of 53 kDa nerve growth factor as studied by a blot and culture technique; Lakshmanan J et al.; A blot and culture technique has been utilized to examine the biological actions of 53,000 Dalton (53 kDa) nerve growth factor (NGF) present in the submaxillary gland (SMG) of adult male and female mice . SMG-proteins were first resolved by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) under reducing conditions and then transferred to a nitrocellulose (NTC) sheet by electroblotting . Specific areas corresponding to 53 kDa NGF were cut and cultured with the rat pheochromocytoma cell line, PC-12 . NTC strips bearing the 53 kDa NGF band stimulated cell flattening and neurite formation in serum containing growth medium and augmented cell survival in serum-free growth medium . Similar results were obtained with NTC strips bearing the 13 kDa NGF band . The results suggest that 53 kDa NGF, like 13 kDa NGF, exerts both tropic and trophic actions on its target neurons.

Neurosci Lett, 1989 May 8, 99(3), 257 - 62
Induction of neurite outgrowth from chick embryonic ganglia explants by activators of protein kinase C; Hsu L et al.; Neurite outgrowth from chick embryonic sensory ganglia explants was induced by activators of protein kinase C and other compounds known to stimulate the hydrolysis of inositol phospholipids . The addition of diacylglycerols, phospholipase C and muscarine chloride to a defined growth medium promoted the outgrowth of dense neurites from ganglia explants which were morphologically distinct from those induced by the phorbol ester TPA . Moreover, these neurite-promoting agents did not enhance non-neuronal cell proliferation and were ineffective in the absence of insulin and/or progesterone.

Biochem Int, 1989 May, 18(5), 913 - 22
Reduced sensitivity of peripheral cells to glucocorticoids in hypercholesterolemia; Shakhov YuA et al.; It has been found that lymphocytes of hypercholesterolemic (HCh) subjects are characterized by a reduced number of glucocorticoid receptors (GcR) as compared with the cells of normolipidemics (N) . Addition of HCh-sera or very low density lipoproteins, or low density lipoproteins isolated both from HCh-sera and N-sera to cultured human skin fibroblasts brought about a fall in the number of GcR in the cells . High density lipoproteins had no effect on GcR level . Dexamethasone was less effective in inhibiting cholesterol synthesis from {14C}acetate in the lymphocytes and fibroblasts with a reduced number of GcR . In the presence of dexamethasone (I x 10(-8)M) in fibroblast growth medium, reduced number of GcR (due to preincubation with very low density lipoproteins) led to a substantial increase in cholesterol synthesis . These findings indicate that the sensitivity of peripheral cells to glucocorticoids is decreased in HCh which might be one of the trigger mechanisms of atherogenesis.

In Vitro Cell Dev Biol, 1989 May, 25(5), 471 - 6
Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes; Merrill GF; A muscle cell strain capable of forming contracting myotubes was isolated from an established rat embryo cell line . The myogenic cells, termed rat myoblast omega or RMo cells, have a diploid complement of chromosomes (n = 42) . In the presence of mitogen-containing growth medium, RMo cells proliferated with a cell generation time of about 12 hours . In mitogen-depleted medium, RMo cells withdrew from the cell cycle and formed myotubes that spontaneously contracted . Differentiated RMo cells produced creatine kinase isozymes in a ratio characteristic of skeletal muscle cells . RMo cells were easy to cultivate . Cells proliferated and differentiated equally well on gelatin-coated or noncoated culture dishes, at clonal or mass culture densities, and in all basal media tested . In most experiments, growth medium consisted of horse serum-containing medium supplemented with either chicken embryo extract or FGF activity; cells proliferated equally well in medium containing unsupplemented calf serum . RMo cells differentiated if growth medium was not replenished regularly . Alternatively, differentiation was induceable by incubation in mitogen-depleted medium consisting of basal medium supplemented either with 10(-6) M insulin, 0.5% serum, or 50% conditioned growth medium . RMo cells were competently transformed with cloned exogenous genes . Because it forms functional myofibrils, the RMo cell line constitutes a useful model system for studying the cell biology and biochemistry of proteins involved in contractile apparatus assembly and muscle disease.

Arch Environ Contam Toxicol, 1989 May-Jun, 18(3), 331 - 5
Analysis and screening for mycotoxins and other secondary metabolites in fungal cultures by thin-layer chromatography and high-performance liquid chromatography; Frisvad JC et al.; Methods for the screening of fungal cultures for toxic secondary metabolites are reviewed . Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are good general analytical methods for secondary metabolites in unpurified extracts . The combination of normal phase TLC using different chemical spray reagents with reversed phase HPLC, using alkylphenone retention indices and diode array detection, is a powerful technique for identifying the individual mycotoxins detected . The results of the screening methods are very dependent of the growth media and incubation conditions and a general method for the detection of Penicillium, Aspergillus, Fusarium, Alternaria and Cladosporium toxins is suggested.

Mol Microbiol, 1989 May, 3(5), 593 - 9
Role of cysteine residues and of metal ions in the regulatory functioning of FNR, the transcriptional regulator of anaerobic respiration in Escherichia coli; Trageser M et al.; FNR, the transcriptional regulator of gene expression of anaerobic respiration in Escherichia coli, contains a cluster of cysteine residues at the amino terminus which resembles the metal-binding domains of metal-binding proteins . It is possible, therefore, (i) that FNR binds metals with the cysteines as ligands and (ii) that this property is related to the regulatory function of FNR . These questions were investigated, with the following results . Approximately 2.4 of the 4 cysteine residues of FNR can be alkylated with iodoacetate in permeabilized aerobic or anaerobic bacteria without the addition of reducing agents . The time required for half-maximal labelling of the cysteines was 50 min in anaerobic bacteria and 6 min in aerobic bacteria . The difference in the reactivity was specific for the cysteines of FNR . These cysteine residues were also highly reactive in anaerobically grown bacteria, when the growth medium contained chelating agents such as 1,10-phenanthroline (15 microM) . The effect of the chelating agents was reversed by an excess of divalent metal ions such as Fe(II) or Cu(II) in the medium . The presence of 1,10-phenanthroline (10 microM) also inhibits the expression of fumarate reductase, an FNR-dependent enzyme . These results suggest that FNR exists in two different forms which differ in terms of the reactivity of their cysteine residues to iodoacetate . The interconversion of both forms appears to be regulated by the availability of O2 and by the binding of metal ions . The two forms of FNR may be involved in the regulation of O2-dependent gene expression.

Indian J Exp Biol, 1989 May, 27(5), 437 - 41
Influence of exogenous thymidine on killing and mutation of Chinese hamster V-79 cells by X-rays, ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine; Ghosh R et al.; V-79 cells when exposed to thymidine (5 micrograms/ml) in growth medium after treatment with X-rays, UV light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), responded differently depending upon the agent . For treatment with X-rays and UV light, only induction of mutation was potentiated, but for MNNG treatment, both killing and mutation induction were potentiated . The increase in killing of MNNG exposed cells could be reversed by simultaneous addition of deoxycytidine with thymidine, but, for all the three mutagenic treatments, enhancement in mutation induction could not be suppressed by deoxycytidine.

Mol Microbiol, 1989 May, 3(5), 601 - 8
FNR-dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR-regulated gene expression; Spiro S et al.; The ndh gene of Escherichia coli which encodes an NADH dehydrogenase contains a putative FNR-binding site in its upstream non-coding region, and its expression has been investigated using an ndh-lacZ fusion . Expression of the fusion was found to be reduced during anaerobic growth, and experiments with hosts containing an fnr mutation and/or a multicopy fnr+ plasmid indicated that the anaerobic repression of the ndh gene is mediated by the FNR protein . Thus FNR can function as an anaerobic repressor as well as an anaerobic transcriptional activator . The results are consistent with the FNR-binding function attributed to the proposed consensus sequence . Using frdA- and ndh-lacZ fusions exhibiting positive and negative regulation by FNR, it was further shown that the depletion of metal ions in growth media with chelating agents mimics oxygen with respect to the activity of FNR . Possible roles for metal ions in the oxygen-sensing pathway associated with FNR function are discussed.

Exp Eye Res, 1989 May, 48(5), 667 - 77
Polyol and vacuole formation in cultured canine lens epithelial cells; Nagata M et al.; Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 mM D-galactose or 30 mM D-glucose . These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 microM sorbinil) . In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 mM mannitol, which is known to enter cells only slowly, or in 30 mM L-galactose, which is not a substrate for aldose reductase . The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 mM D-galactose and increased in both number and size with time . These vacuoles had a unique fine structure . They did not result from swelling of mitochondria or other cell organelles . As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules . The proliferation pattern of cells incubated with 30 mM D-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium . Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

Jpn J Cancer Res, 1989 May, 80(5), 464 - 8
High induction of poly(ADP-ribose) polymerase activity in bleomycin-resistant HeLa cells; Urade M et al.; Poly(ADP-ribose) polymerase activity was measured in bleomycin (BLM)-resistant HeLa (HeLa-BLMr) and the parental HeLa cells after BLM treatment . HeLa-BLMr cells, which had been subcultured in growth medium containing 1 micrograms/ml of BLM, showed a 3.75-fold higher enzyme activity than did HeLa cells, but this activity was decreased to the same level as that of HeLa cells after 48 h of BLM-free cultivation . When HeLa and HeLa-BLMr cells after a 48-h cultivation in BLM-free growth medium were treated with BLM, the enzyme activity was induced at a higher level (2.5-3.8 times) in HeLa-BLMr than in HeLa cells and was inhibited markedly in HeLa-BLMr and slightly in HeLa cells by nicotinamide, an inhibitor of this enzyme . The BLM-induced cell killing by nicotinamide was highly potentiated (about 18 times) in HeLa-BLMr as compared to HeLa cells.

J Cell Biol, 1989 May, 108(5), 1967 - 77
A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants; Pennell RI et al.; We have identified a family of abundant peripheral plasma membrane glycoproteins that is unique to flowering plants . They are identified by a monoclonal antibody, MAC 207, that recognizes an epitope containing L-arabinose and D-glucuronic acid . Immunofluorescence and immunogold labeling studies locate the MAC 207 epitope to the outer surface of the plasma membrane both in protoplasts and in intact tissues . In some cells MAC 207 also binds to the vacuolar membrane, probably reflecting the movement of the plasma membrane glycoproteins in the endocytic pathway . The epitope recognized by MAC 207 is also present on a distinct soluble proteoglycan secreted into the growth medium by carrot (Daucus carota) suspension culture cells . Biochemical evidence identifies this neutral proteoglycan as a member of the large class of arabinogalactan proteins (AGPs), and suggests a structural relationship between it and the plasma membrane glycoproteins . AGPs have the property of binding to beta-glycans, and we therefore propose that one function of the AGP-related, plasma membrane-associated glycoproteins may be to act as cell surface attachment sites for cell wall matrix polysaccharides.

Thromb Haemost, 1989 Apr 25, 61(2), 314 - 7
Effect of the lupus anticoagulant on endothelial fibrinolytic activity in vitro; Francis RB Jr et al.; We investigated the effect of plasma and serum from 10 subjects with the lupus anticoagulant and thrombosis and 9 normal subjects on the secretion of tissue-type plasminogen activator (t-PA) and its rapid inhibitor (type 1 plasminogen activator inhibitor, or PAI-1) by cultured human endothelial cells . Confluent monolayers of human umbilical vein endothelial cells were incubated for 48 hours with plasma or serum diluted ten-fold in serum-free endothelial cell growth medium, and the secretion of t-PA and PAI-1 measured by enzyme-linked immunosorbent assay . No consistent differences in mean t-PA and PAI-1 release were found between cells exposed to normal plasma or serum and plasma or serum from subjects with the lupus anticoagulant and thrombosis . No plasma or serum sample produced consistent inhibition of t-PA release or stimulation of PAI-1 release (defined as t-PA levels less than the mean minus two standard deviations for normal subjects, and PAI-1 levels greater than the mean plus two standard deviations for normal subjects, respectively) . These findings do not support a role for altered endothelial fibrinolytic activity in the pathogenesis of thrombosis in subjects with the lupus anticoagulant, and are consistent with previous observations that these subjects have normal fibrinolytic activity in vivo.

J Neurochem, 1989 Apr, 52(4), 1156 - 61
Neurofilament phosphorylation in cultured bovine adrenal chromaffin cells is stimulated by phorbol ester; Georges E et al.; Primary cultures of bovine adrenal chromaffin cells contain neurofilament proteins that are hypophosphorylated . When the cells were grown in medium containing 32Pi and 0.1 microM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), 32P-labelling of the three neurofilament subunits was increased 6- to 20-fold relative to controls, the highest level of stimulation occurring for the mid-sized subunit . Addition of the protease inhibitor leupeptin to the growth medium had no effect on TPA-stimulated phosphorylation . The increased 32P incorporation was accompanied by a marked reduction in the gel electrophoretic mobilities of the two largest subunits . The augmented phosphorylation was observed 10 min after addition of TPA to a concentration of 0.1 microM or after 1 h of incubation in the presence of 0.01 microM TPA . One-dimensional peptide mapping and phosphoamino acid analysis indicated that TPA stimulated the phosphorylation of seryl residues at new sites in the mid-sized subunit . All of the latter subunit contained in the cytoskeletal fraction of chromaffin cells was converted to a more highly phosphorylated state after the cells were grown in the presence of TPA for 1 h.

J Oral Pathol Med, 1989 Apr, 18(4), 214 - 9
Cell culture and characterization of human minor salivary gland duct cells; Kurth BE et al.; In order to facilitate studies on human salivary glands, a method was developed for the culture of minor salivary gland duct cells from tissues obtained from oral surgery protocols . Minor salivary glands were isolated from such tissues, and a serum-free growth medium was developed which supported the growth of the ductal component of these glands . The ductal origin of these cells was confirmed through immunohistochemical localization of replicating nuclei through incorporation of BrdU . The presence of epidermal keratin in replicating cells and the absence of smooth muscle myosin further substantiated the ductal origin of cells . Using normal growth medium calcium concentrations (1.05 mM), these cells produced a keratinized multilayer of cells unable to undergo routine subculture procedures . A reduction in calcium ion concentration to 0.1 mM resulted in a cell monolayer, without evidence of terminal keratinization, which could undergo at least eight serial passages (1:3 ratio) under cell culture conditions . It is advanced that these minor salivary gland duct cell cultures will be of use to those studying diseases and disorders of the salivary glands.

Gen Comp Endocrinol, 1989 Apr, 74(1), 32 - 44
Effects of precocene analogs on the nematode Caenorhabditis remanei (var . Bangaloreiensis) . II . Competitions with a juvenile hormone analog (Methoprene); Fodor A et al.; Fourteen 7-alkoxy-2,2-dimethychromenes were synthetized and studied in JH competition experiments: precocenes (Ps) PI and PII, and synthetic analogs (PAs) including (i) three with both antiallatal and P-like activities: 7-ethoxy-PII (7-EPII); 7-(prop-2-ynyloxy)-2,-2-dimethylchromene (PPI); and 6-methoxy-7-(prop-2-ynyloxy)-2,2-dimethylchromene (PPIII); (ii) six without antiallatal activity, exerting P-like activity in nematodes; and (iii) three without either antiallatal or P-like activity, but with a strong nematocidal effect . Within the dose range 8-1000 micrograms/ml, different concentrations of each PA were applied to nematode growth medium which did or did not contain 1000 micrograms methoprene (a juvenile hormone analog JHA)/ml . Plates inoculated with Caenorhabditis embryos were incubated and scored for developmentally affected survivors . The JHA did not compete with any PA mentioned as (iii) . It competed moderately with some nonantiallatal PAs (8-Me-PPI, 8-MeO-PPI, and 3,4-diCl-PPI) with strong P-like and nematocidal activities . The JHA competed most efficiently with all Ps, antiallatal PAs, and two nonantiallatal PAs (PPII and thio-PI) which exerted severe P-like activities in nematodes . Parameters assumed to be indicators of the P-like (rather than nematocidal) activity of the PAs proved more sensitive to the JHA than those of nematocidal activity . Whether the JH-compensable P-like activity of some PAs can be regarded as a real anti-JH action needs further clarification.

J Neurol Sci, 1989 Apr, 90(2), 167 - 77
Myosin heavy chain expression in human muscle cocultured with mouse spinal cord; Ecob-Prince M et al.; Monoclonal antibodies which specifically recognise the neonatal or adult fast (type 2A and 2B) or slow myosin heavy chain (MHC) were used to investigate the myosin composition of human muscle which had regenerated in culture in the presence or absence of embryonic mouse spinal cord tissue . Adult fast MHC was found in an average of 75% of the cultured fibres, irrespective of the time in culture, the source of the muscle, the presence of neurones or modifications to the growth medium . It was not found alone but was always in association with the neonatal and/or slow myosin isoforms . The expression of fast MHC in human muscle therefore differs from that in mouse muscle in this culture system (Ecob-Prince et al . 1986), but is still strong evidence for the development of at least one aspect of an adult phenotype in culture.

Am J Obstet Gynecol, 1989 Apr, 160(4), 954 - 60
Purified human early pregnancy factor from preimplantation embryo possesses immunosuppresive properties; Bose R et al.; This study was undertaken to determine whether early pregnancy factor secreted by preimplantation embryos has immunosuppressive properties . Human early pregnancy factor was purified from embryo growth media of in vitro fertilized ova with ion-exchange and gel filtration chromatography . During each step of purification the fractions were tested for (1) early pregnancy factor activity with the rosette inhibition assay, (2) immunosuppressive properties with a concanavalin A-stimulated lymphocyte proliferation assay, and (3) purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Results indicate that (1) human early pregnancy factor has a basic molecular weight of 14 kd, (2) early pregnancy factor has immunosuppressive activity, (3) polymers of early pregnancy factor also appear to be present in the embryo growth media, and (4) immunosuppressive factors other than early pregnancy factor are also secreted by preimplantation human embryos . Early pregnancy factor and other factor(s) produced by the preimplantation embryo may play a role in suppressing maternal cellular immune responses, thereby preventing maternal rejection of the embryo.

Yeast, 1989 Apr, 5 Spec No, S447 - 51
Identity of soluble thiamine-binding protein with thiamine repressible acid phosphatase in Saccharomyces cerevisiae; Nosaka K et al.; Two secretory glycoproteins of S . cerevisiae, a soluble thiamine-binding protein and a thiamine-repressible acid phosphatase, were shown to be repressed to a similar extent by excess thiamine in the growth medium . Thiamine-repressible acid phosphatase was co-purified throughout the purification of the soluble thiamine-binding protein . Purified and deglycosylated soluble thiamine-binding proteins exhibited both thiamine-binding and acid phosphatase activities on non-denaturing polyacrylamide gel electrophoresis . Heat treatment of the purified soluble thiamine-binding protein caused a decrease in both activities with a similar inactivation profile . Two thiamine-repressible acid phosphatase-defective mutants isolated were found to be also defective in soluble thiamine-binding activity . The uptake of {14C}thiamine phosphate esters, such as thiamine monophosphate and thiamine pyrophsphate, was remarkably impaired in the mutant cells, whereas the uptake of {14C}thiamine by the mutant was almost the same with that by the parent strain . From these results, it was concluded that the soluble thiamine-binding protein is identical to the thiamine-repressible acid phosphatase in S . cerevisiae, which is involved in the hydrolysis of exogenous thiamine phosphate esters in the periplasmic space prior to the uptake of their thiamine moiety by yeast cells.

Mol Gen Genet, 1989 Apr, 216(2-3), 469 - 74
Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase; Kashlev MV et al.; Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase beta-subunit gene in the chromosome and a rifampicin resistant beta-subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit . A major portion of the overproduced subunit is found in an insoluble form . Conditions known to induce the heat shock proteins (hsps), e.g . elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne beta-subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant . Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps . We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with beta-subunit aggregation.

Indian J Biochem Biophys, 1989 Apr, 26(2), 87 - 91
Effect of dimethyl sulphoxide on mitochondrial biogenesis in regenerating rat liver and Saccharomyces cerevisiae; Desai SD et al.; Effect of dimethyl sulphoxide (DMSO) on mitochondrial biogenesis in regenerating rat liver and cells of Saccharomyces cerevisiae during aerobiosis has been studied by monitoring the cytochrome oxidase activity . A single dose of DMSO (275 mg/100-125 g body wt) to normal rats stimulated cytochrome oxidase activity in liver mitochondria while the same dose to partial hepatectomized rats inhibited the enzyme activity . Administration of low dose of DMSO (92 mg/100-125 g body wt) to partial hepatectomized rats did not alter the enzyme activity . Anaerobic cells of S . cerevisiae on aerobiosis for 2 hr attained cytochrome oxidase activity level on par with aerobic cells . Inclusion of DMSO (275 mg/100 ml) in the growth medium of S . cerevisiae during respiratory adaptation exerted partial inhibitory effect on the formation of cytochrome oxidase at 2 hr period, while the 10-fold concentration inhibited the enzyme formation completely . However, the inhibitory effect of DMSO on enzyme formation was abolished on prolonged growth (18 hr and above), while these doses had no influence on cytochrome oxidase in aerobic cells of S . cerevisiae . The results imply that DMSO may be exerting its effect on the assembly of subunits into active enzyme complex during mitochondrial biogenesis.

J Bacteriol, 1989 Apr, 171(4), 2124 - 7
Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli; Kilstrup M et al.; Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis . After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated . One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine . The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD) . The mutation was mapped by P1 transduction at 36 min on the E . coli linkage map . A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation . By comparing the restriction maps of the cloned fragment and the E . coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min).

Cancer Lett, 1989 Apr, 45(1), 43 - 8
Effect of dietary brussels sprouts with increased selenium content on mammary carcinogenesis in the rat; Stoewsand GS et al.; Brussels sprouts (Brassica oleracea, L; Jade cross E, hybrid cultivar) were cultivated with inorganic selenium added to the plant growth medium . Sprague-Dawley, female, weanling rats were divided into groups and fed 20% brussels sprouts diets containing either 0.03, 0.58, 1.29, or 6.71 ppm of selenium naturally occurring in the sprouts . These diets were fed 2 weeks before and 2 weeks after a single dose of 7,12-dimethylbenz{a}anthracene (DMBA), and the rats were then placed on a low selenium basal diet for an additional 25 weeks . All brussels sprouts diets reduced the incidence of DMBA-induced mammary carcinogenesis . Increased dietary levels of naturally occurring selenium did not further depress mammary tumorigenesis . The time periods of selenium feeding may have been too brief to observe any additional tumor reductions.

Cancer Res, 1989 Mar 15, 49(6), 1509 - 14
Effects of 5-hydroxymethyluracil and 3-aminobenzamide on the repair and toxicity of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells; Boorstein RJ et al.; 5-Hydroxymethyl-2'-deoxyuridine (HmdUrd), a cytotoxic analogue of thymidine, has been proposed for use as an anticancer agent . HmdUrd is incorporated into DNA and then removed at a rate of 30-40% per day . The removal of HmdUrd from DNA has been attributed to the action of 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase) . We demonstrated the release of {3H}HmUra into the growth medium of V79 Chinese hamster cells that had incorporated {3H}HmdUrd into their DNA . The amount of {3H}HmUra recovered from the growth medium was equal to the amount of {3H}HmdUrd lost from DNA . These experiments confirmed that the initial step of repair of HmUra in DNA was mediated by DNA glycosylase activity . A combination of HmUra and HmdUrd resulted in increased uptake of HmdUrd by cells and increased cytotoxicity . The increased incorporation of HmdUrd into DNA was not due to inhibition of repair . 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, was cytotoxic to cells which incorporated and repaired HmdUrd . The extent of toxicity was directly related to the number of HmUra residues in DNA . HeLa cells, known to be resistant to the toxic effects of HmdUrd, do not incorporate HmdUrd into their DNA . HeLa cells were resistant to the toxic effects of 3-aminobenzamide, confirming that the absence of HmdUrd in their DNA was not due to an accelerated rate of repair . These experiments indicate that the potential therapeutic antineoplastic properties of HmdUrd may be enhanced by using HmUra to increase the incorporation of HmdUrd into DNA and 3-aminobenzamide to interfere with repair of HmUra in DNA.

Gene, 1989 Mar 15, 76(1), 27 - 39
Construction of an EBNA-producing line of well-differentiated human hepatoma cells and of appropriate Epstein-Barr virus-based shuttle vectors; Lutfalla G et al.; Using cloned Epstein-Barr nuclear antigen 1 (EBNA) and oriP elements from the Epstein-Barr virus (EBV) in conjunction with liver-specific growth media, we have constructed an EBNA-producing line of well-differentiated human hepatoma cells (Hep-EBNA-2) and appropriate EBV-oriP vectors . These vectors, pBEDC1 and pBEUG1, were maintained as free extrachromosomal elements only in cells that expressed the trans-acting EBNA protein . They were readily rescued from transfected Hep-EBNA-2 cells upon transformation of recA- Escherichia coli with cellular low-Mr DNA . They are true shuttle vectors in that they can propagate as free closed circular elements in both human Hep-EBNA-2 cells and E . coli . Finally, we have demonstrated the vector capability of our shuttle system by inserting into the SV40 expression cassette of pBEUG1 a large full-length cDNA encoding coagulation factor VIII . Our data clearly show that EBV-oriP episomes are able to stably propagate in an hepatic background and that neither high levels of EBNA protein nor multiple copy episomes significantly interfere with the expression of the set of hepatic functions that have been analyzed . These results are discussed in terms of gene amplification and cloning of genes that program liver differentiation.

Biochim Biophys Acta, 1989 Mar 14, 1002(1), 20 - 7
Correlation of the dispersion state of pyrene cerebroside sulfate and its uptake and degradation by cultured cells; Viani P et al.; This study aimed at increasing the efficiency and shortening the time required for administering cerebroside sulfate to cultured cells . For this purpose several modes of dispersion of a fluorescent derivative of cerebroside sulfate (sulfatide), in which the natural fatty acid has been replaced by pyrenedodecanoic acid (P12), were incubated with the cells . This fluorescent derivative of cerebroside sulfate (P12-CS) was introduced into the growth medium of the cells using the three following modes of dispersion: (1) P12-CS was dissolved in dimethylsulfoxide and added to the medium, (2) it was precomplexed with serum albumin or (3) incorporated into small, unilamellar vesicles (SUV) of phosphatidylcholine . With each of these respective modes of dispersion, the P12-CS was incubated for periods up to 48 h with cultured lymphoblasts or fibroblasts . Uptake by the cells could be determined by recording directly the cell-associated fluorescence, using a suspension of washed intact cells . The cell lipids were subsequently extracted with mixtures of chloroform/methanol and their fluorescence recorded . When related to the incubation time, uptake of P12-CS by the cells increased continuously using each of the above dispersions . The appearance of fluorescence at 475 nm ('excimer') and the ratio of this to the monomolecular fluorescence at 378 nm ('E/M') could be used as a measure for the presence of the internalized P12-CS in aggregated or fully dispersed states . These values (i.e., E/M), recorded on the suspensions of intact cells were rather high using the aqueous dispersions, intermediate values were observed using the SUV and rather low E/M values (0.5 or less) were observed using the preformed albumin-(P12-CS) complexes . Increasing the mole ratio of albumin to P12-CS (i.e., from 1:2 to 2:1 m/m), decreased the quantity of sulfatide which was taken up by the cells but also further decreased the E/M ratio, suggesting a fully dispersed state of the pyrene lipid within the cell . This indicated that, using an optimal albumin to P12-CS ratio of 1-2 (or its equivalent values in fetal calf serum) permitted an influx of single molecules of P12-CS into the cells . After 48 h, about 50% of the fluorescence of skin fibroblasts was found in metabolic degradation products of P12-CS . The parallel value for fibroblasts derived from a patient with metachromatic leukodystrophy was only about 5% . Appearance of the excimeric emission of a dispersion of P12-CS in water permitted estimation of its critical micellar concentration as being 7.5 x 10(-7) M.

Mikrobiologiia, 1989 Mar-Apr, 58(2), 202 - 5
{The effect of yeast autolysates on the level and distribution of free amino acids in yeast cells of the Candida genus}; Gusel'nikova TV et al.; The work was aimed at studying the effect of yeast autolysate on the content and subcellular distribution of free amino acids in yeast cells . The overall pool of free amino acids decreased 1.5-2 times when mineral nitrogen in the growth medium was substituted either completely or partly by yeast autolysate . As was shown using the technique of differential extraction, the vacuolar pool of the cell is mainly responsible for the decrease in the content of free amino acids.

Immunol Lett, 1989 Mar, 20(4), 261 - 7
Human embryo associated immunosuppressor factor(s) from pre- and post-implantation stages share some similarities; Bose R; The present study demonstrates that embryo associated immunosuppressive factor(s) (EASF) secreted by the human embryo at pre- and post-implantation stages share some similarities . Human EASF was partially purified from embryo growth media of in vitro fertilized ova and from first trimester pregnancy sera . Non-pregnancy sera were fractionated in parallel . During each step of purification the fractions were tested for immunosuppressive properties using concanavalin A-stimulated lymphocyte proliferation assay . Analysis of EASF positive fractions on SDS-PAGE identified 14 kDa and 24 kDa molecules in embryo growth media and pregnancy sera . No such molecules were found in control sera, suggesting that these factors are embryo associated . The relationship of pre- and post-implantation EASF was also analyzed by EASF binding assay using murine anti-EASF antibody, which was raised against EASF isolated from embryo growth media . Results show that murine antibody bound to EASF purified from pregnancy sera, but not to identical fractions from control sera, indicating that these post-implantation EASF possess some similarity with pre-implantation EASF . Results also indicate that a species of suppressor factors present in embryo growth media and pregnancy sera were unique for their origin . Presence of these three EASFs at various stages of gestation may play a role in suppressing maternal cellular immune responses thereby preventing maternal rejection of the embryo.

Steroids, 1989 Mar-May, 53(3-5), 607 - 23
Regulation by heme of sterol uptake in Saccharomyces cerevisiae; Shinabarger DL et al.; The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols . Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth . The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216 . Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin . Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake . These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.

Int J Radiat Oncol Biol Phys, 1989 Mar, 16(3), 707 - 14
The influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells; Osmak M et al.; Cell response to irradiation depends on many micro-environmental and intracellular factors . It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle . They also could be involved in cell response to irradiation . In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied . Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth . Immediately after irradiation cathepsin B or leupeptin were added to the growth medium . Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction . Mutation frequency was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG) . Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals . The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants . If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency . The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases . As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities.

Int J Radiat Biol, 1989 Mar, 55(3), 365 - 84
Similar lethal effect in mammalian cells for two radioisotopes of copper with different decay schemes, 64Cu and 67Cu; Apelgot S et al.; The decays of 64Cu incorporated in human malignant (A549) or monkey nonmalignant (CVI) cells lead to cell death . When plotted as a function of the radioactivity introduced in the growth medium (microCi/ml at t = 0), the residual colony-forming capability decreases exponentially . The slope of the corresponding curve is steeper for A549 than for CV1 cells . Different data show that the cellular lethal event is a consequence of 64Cu transmutation and not of the irradiation by the simultaneously emitted beta- and beta+ particles . Liquid holding results show that the lethal event is irreparable . The decays of 67Cu, another radioisotope of copper, lead to cell death with the same exponential survival curve and the same lethal efficiency as for 64Cu, in spite of their different decay schemes . The lethal efficiency of both copper isotopes is close to that of 125I utilized in the form of iododeoxyuridine under the same experimental conditions as 64Cu and 67Cu . The high lethal efficiency of radioactive copper transmutations raises questions about the role in DNA functioning of copper atoms known to be present in trace amounts in this macromolecule . The lethal consequence of radioactive copper transmutations suggests that the copper atoms bound to DNA are essential for cellular functioning.

Mol Cell Biol, 1989 Mar, 9(3), 1233 - 42
Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells; Kaufman RJ et al.; In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide . This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF) . Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF . Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium . In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated . vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells . Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm . In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF . These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

Am Rev Respir Dis, 1989 Mar, 139(3), 715 - 20
The effect of parainfluenza 3 infection on guinea pig basophil and lung mast cell histamine release; Graziano FM et al.; The guinea pig infected with parainfluenza 3 (P-3) has provided an animal model to study mechanisms of virus-induced asthma and airway hyperreactivity . Evidence to identify the mechanisms by which P-3 infection enhances airway hyperreactivity, however, has not been established . The present study evaluated the effect of P-3 infection on histamine release (HR) from isolated peripheral blood guinea pig basophils and lung mast cells . Basophil HR was determined in animals made basophilic with 13 daily intraperitoneal injections of sheep blood . On Day 10 of the sensitization, blood was obtained from all animals, and leukocyte HR was determined to sheep gammaglobulin (SG), which served as the secretagogue . After these baseline studies, the animals were separated into two groups; one was insufflated with P-3 virus and the other with growth medium that did not contain virus . Four days later, animals were killed, basophils and lung mast cells were isolated, HR was determined, and lung tissue was cultured for the presence of virus . In basophils isolated from animals proven to be infected with P-3 virus, we found significantly more histamine released in response to the lower doses of SG and a significant shift in the -log EC50 dose of this antigen causing HR in these cells . Experiments conducted with the calcium ionophore A23187 as the basophil secretagogue in the same experimental design did not demonstrate similar HR findings . Furthermore, a defect in calcium disposition did not account for the enhanced HR from basophils isolated from infected animals . HR from pulmonary mast cells was similar in cells isolated from P-3-infected and control insufflated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1989 Feb 25, 264(6), 3514 - 23
Characterization of the major heparan sulfate proteoglycan secreted by bovine aortic endothelial cells in culture . Homology to the large molecular weight molecule of basement membranes; Saku T et al.; To determine the precise architecture and functional characteristics of the subendothelial basal lamina, detailed information of the molecules contained in this structure is required . To this end, we have studied low passage bovine aortic endothelial cells and have isolated the major heparan sulfate-containing proteoglycan from the growth medium of the cells maintained under static culture conditions . This large macromolecule consists of a core protein approximately 500,000 daltons in mass and two to three glycan side chains as revealed by carbon/platinum rotary shadow casting . Specific antibodies raised by immunization of rabbits with the native or deglycosylated bovine molecule could be isolated from an immunoadsorption column prepared with a preparation isolated from the murine Engelbreth-Holm-Swarm tumor . The antibodies purified by immunoaffinity react with basement membranes of blood vessels, lung, liver, or skin, and this reactivity is indistinguishable, at least for the organs studied, from the reactivity of antibodies specific for the Engelbreth-Holm-Swarm tumor-derived high molecular weight heparan sulfate proteoglycan isolated previously . Immunoelectron microscopy of frozen ultrathin tissue sections from the kidney indicates localization of the epitope(s) also in the basement membranes of the renal glomeruli and tubuli . The close structural relationship and homology between the aortic endothelial cell product can be demonstrated even more convincingly by two-dimensional peptide mapping procedures . The peptide patterns from the bovine and mouse products of approximately 500 kDa are nearly indistinguishable . Maps of polypeptides of molecular masses ranging from 400 to 150 kDa, which are found in the bovine as well mouse tumor preparation, are clearly related to each other and suggest that this proteoglycan is quite sensitive to degradation by tissue proteases . Thus the data presented here strongly suggest that the large proteoglycan previously isolated and described as a tumor cell product can be produced by normal cells.

FEBS Lett, 1989 Feb 13, 244(1), 11 - 4
Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum; Shoun H et al.; Both soluble and microsomal fractions of Fusarium oxysporum contain cytochrome P-450(P-450) . We report here that the P-450 in the soluble fraction was induced only when nitrate or nitrite was added to the growth medium, whereas the microsomal P-450 was synthesized regardless of the medium compositions . The reduced-CO complex of the soluble P-450 exhibited an absorption spectrum that is different from that of the microsomal counterpart . These results indicate that the soluble P-450 is distinct from the microsomal species and suggest a novel function for the former P-450.

Anal Biochem, 1989 Feb 1, 176(2), 303 - 6
Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae); McEntee CM et al.; High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures . The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells . Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase . Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded . Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.

J Cell Biochem, 1989 Feb, 39(2), 153 - 66
Retroviruses expressing different levels of the normal epidermal growth factor receptor: biological properties and new bioassay; Velu TJ et al.; Two retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFR cDNA into two different retroviral vectors . One DNA (pCO11-EGFR-neo) also contained a linked selectable marker gene (neoR) . The other (pCO12-EGFR) only expresses hEGFR . When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium . Compared with pCO11-EGFR-neo, pCO12-EGFR induced EGF-dependent transformation 2-5 times more efficiently and expressed higher numbers of receptors (4 x 10(5) vs . 1 x 10(5) EGF receptors per cell) . The results indicate that transforming potential is directly related to the number of EGF receptors . In defined, serum-free medium that contained only very low concentrations of insulin (0.6 microgram/ml) and transferrin (0.6 micrograms/ml), hEGFR-virus infected cells were able to grow with EGF as the only growth factor . Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth . NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus . The dose-dependent growth response to EGF of infected NR6 cells grown in serum-free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type alpha (TGF alpha) . This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg-100 ng/ml) . Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF specificity of growth stimulation.

Ecotoxicol Environ Saf, 1989 Feb, 17(1), 94 - 104
Impact of chromium and tin on a nitrogen-fixing cyanobacterium Anabaena doliolum: interaction with bivalent cations; Rai LC et al.; The toxicity of chromium and tin on growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of Anabaena doliolum, and its interaction with bivalent cations, viz . Ca2+, Mg2+, Mn2+, Ni2+, Co2+, and Zn2+, has been studied . Some interacting cations, viz . Ca, Mg, and Mn, substantially antagonized the toxic effects of chromium and tin with reference to growth and nutrient (NO3- and NH4+) uptake in the hierarchical sequence Ca greater than Mg greater than Mn, whereas the sequence of hierarchy was Mn greater than Mg greater than Ca for nitrate reductase and glutamine synthetase activity of A . doliolum . A synergistically inhibitory pattern of interaction was noted for all the parameters, viz . growth, uptake of NO3- and NH4+, nitrate reductase and glutamine synthetase activity of A . doliolum, when Ni, Co, and Zn were used in combination with test metals in the growth medium . These bivalent cations followed the synergistic inhibition sequence Ni greater than Co greater than Zn and potentiated the toxicity of test metals in the N2-fixing cyanobacterium under study.

FASEB J, 1989 Feb, 3(2), 163 - 8
Elimination of permeability mutants from selections for drug resistance in mammalian cells; Schibler MJ et al.; Chinese hamster ovary (CHO) cells exhibit increased sensitivity to a wide variety of microtubule inhibitory drugs when verapamil is present in the growth medium . The extent of this increased sensitivity is drug specific: some drugs such as taxol and vinblastine respond greatly to the presence of verapamil, whereas other drugs such as griseofulvin respond very poorly . For the majority of drugs examined, however, a 2- to 10-fold increase in drug sensitivity is observed in the presence of verapamil at 5 micrograms/ml . The effects of verapamil are even more dramatic when drug-resistant mutant cells with a presumed alteration in membrane permeability are examined . In the presence of appropriate levels of verapamil, these mutants demonstrate a level of drug sensitivity comparable to that of the wild-type parental cells . Drug-resistant cells from similar selections but with well-defined alterations in alpha- or beta-tubulin and no evidence of alterations in membrane permeability, however, continue to exhibit increased resistance to the selecting drug even in the presence of verapamil . These studies support the conclusion that verapamil affects the membrane permeability to or transport of a wide variety of hydrophobic drugs . In addition, we have used this information to devise selections that virtually eliminate the isolation of drug-resistant permeability mutants . This methodology should be generally applicable to genetic studies of drug action that are complicated by the isolation of large numbers of mutants with permeability alterations.

Proc Natl Acad Sci U S A, 1989 Feb, 86(4), 1362 - 6
Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens; Keydar I et al.; We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D . This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients . No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin . The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients . A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay . The results showed that the antigen was detectable in 91% of all breast tumors tested . No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas . Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen . Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.

Biochemistry, 1989 Jan 24, 28(2), 516 - 21
13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans; Pust S et al.; The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques . The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells . In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment . The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment . The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups . The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly . It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor . On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system . In the dithionite-reduced enzyme, the ring system is bent at the N(5) position . Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group . In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1989 Jan 1, 257(1), 173 - 82
Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts . Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells; Higgins PJ et al.; A 52 kDa protein (p52) was identified, using differential extraction and electrophoretic criteria, as a major extracellular and substrate-associated component of normal rat kidney (NRK) fibroblasts . Cells transformed with Kirsten murine sarcoma virus (KNRK cells) did not express p52 constitutively, but were inducible for both p52 production and its substrate association during culture in sodium butyrate (NaB)-supplemented growth medium . Comparative analysis of the relative molecular mass, subcellular distribution, and isoelectric complexity (five variants ranging in pI from 5.4 to 6.2) of the 52 kDa species constitutively and inducibly expressed by NRK and KNRK/NaB cells respectively, indicated that they were, indeed, the same protein . p52 selectively localized to cellular fractions enriched in substrate focal contact sites and associated ventral undersurface components . NaB induction of p52 in KNRK cells occurred before cell spreading; other polar compounds, such as dimethyl sulphoxide, which did not induce KNRK cell spreading, similarly failed to elicit p52 production . p52 accumulated more rapidly in (and was quickly released from) the focal-contact-enriched protein fraction of NRK cells compared with its time course of appearance in the medium . These data collectively suggest that p52 is one of a relatively small number of proteins the synthesis of which is either involved in determination of cell shape or regulated as a consequence of cell-shape changes.

J Med, 1989, 20(3-4), 193 - 207
In vitro growth and hemopoietic differentiation of mouse bone marrow-derived adherent stromal cells in long-term culture: formation of spheroidal bodies mimicking hemopoietic anlagen; Islam A et al.; A novel method is reported for in vitro growth and hemopoietic differentiation of mouse bone marrow-derived adherent stromal cells in long-term marrow cultures . The cultures were done in 50 ml conical tubes with cell suspension in 10 ml of growth medium . The first stage was to form a layer of adherent stromal cells to extend over the entire area of the tube bathed by the medium . The second stage was to develop small spheroidal bodies (1 to 3 mm in diameter) firmly anchored to the substratum of adherent stromal cells . The spheroidal bodies consisted of an outer layer of stromal (primitive mesenchymal) cells, and an inner core of mostly round and primitive hemopoietic cells . Some degree of differentiation into hemopoietic cell lines was also seen in the central core of some spheroidal bodies . These findings show similarities between the spheroidal body formation and the early events of hemopoiesis in human fetal bone marrow, and suggest that the bone marrow stroma cultured in vitro is multipotential and capable of modulation and transformation into stromal and hemopoietic precursor cells.

Dev Biol Stand, 1989, 70, 101 - 7
Are continuous cell lines safe as substrates for human drugs and biologics? A case study with human growth hormone; Carter MJ et al.; The development of a genetically engineered human growth hormone (hGH) preparation is described . The cell line used to produce hGH is a C127 mouse cell transformed with a bovine papilloma virus (BPV) vector . Master, Extended Production, and Working Cell Banks were established . Product is produced by culturing in growth medium, and purified using a series of chromatography and ultrafiltration steps . Extensive testing of cell banks, and bulk and finished product reveals that the cells used to produce hGH are phenotypically and genotypically stable and are free from contamination with adventitious agents . Levels of residual host cell DNA are well below accepted guidelines . Clinical data gathered in multicenter trials confirm the efficacy and safety of the product in the treatment of classic growth hormone deficiency . The regulatory history of human growth hormone in the U.S . is reviewed.

J Cancer Res Clin Oncol, 1989, 115(3), 269 - 75
Uptake and release of iodine-labelled m-iodobenzylguanidine in a neuroblastoma cell culture system and its importance in neuroblastoma therapy; Paffenholz V et al.; A neuroblastoma cell line was established from bone marrow of a patient in stage IV of the disease and used as a model system in order to elaborate experimental data of importance in neuroblastoma therapy, such as cell-drug interactions, the mode of uptake and conditions for storage and release . m-Iodo benzylguanidine (MIBG) is rapidly taken up from culture medium, giving high concentrations of cell-bound radioactivity reaching a maximum level 4 h after the addition of the compound . A removal of the radiopharmacon from the culture medium causes a dramatic loss of cell-associated radioactivity, suggesting that neuroblastoma cells are not able to retain MIBG in a drug-free environment . Replacement of labelled by unlabelled MIBG prevents a similar release and maintains high levels of cellular radioactivity . Variations of cell culture conditions only result in minor changes of uptake rates, whereas a pretreatment with drugs used in neuroblastoma chemotherapy harms the cells extensively: even after a short-term exposure the cells lose the capacity for MIGB uptake and fail to recover within a long period of incubation in growth medium . The importance of our results is discussed, leading to the following suggestions: 1 . The performance of radiotherapy with labelled MIBG is recommended prior to the chemotherapy protocols . 2 . Therapeutically effective radioactivity in tumor tissues may be maintained by the additional infusion of unlabelled MIGB.

Exp Gerontol, 1989, 24(3), 251 - 64
Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions; Hosono R et al.; The nematode Caenorhabditis elegans was cultured monoxenically with E . coli as a food source and the influence of the bacterial growth conditions on the life span was studied . When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematode's life span tended to be prolonged without a marked effect on postembryonic development . The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C . elegans) rather than during the larval to young adult stage . Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.

Environ Mol Mutagen, 1989, 13(3), 253 - 62
Toxic effects of methyl methanesulfonate (MMS) on activated macrophages from chickens; Qureshi MA et al.; Adherent peritoneal exudate cells rich in macrophages were harvested from Cornell K-strain chickens 42 hr after i.p . stimulation with Sephadex G-50 . Glass-adherent monolayers were obtained on coverslips and subjected to in vitro exposure to methyl methanesulfonate (MMS) at various doses for 1 hr . Solvent (0.17% ethanol final concentration) and sham (RPMI 1640 growth media) exposures were also performed . At selected times after exposure, the macrophages were analyzed for cell viability, adherence, DNA damage, and functional activity . Although MMS doses of 5 x 10(-3) M and 1 x 10(-3) M concentrations resulted in significant cytoxicity, 2 x 10(-4) M had no significant cytotoxic effect . However, this exposure resulted in DNA damage as measured by alkaline elution . Concomitant with the DNA damage was a significant decrease in the phagocytic activity of macrophages . Repair of MMS-induced DNA lesions in macrophages was indicated by a normal DNA alkaline elution profile 10 hr postrecovery . Functional activity of cells also returned to normal levels . In contrast, the incidence of Fc receptor-positive cells detected by rosetting increased immediately after MMS exposure, and phagocytosis of opsonized SRBCs was not affected by 2 x 10(-4) M MMS treatment . Similarly, MMS treatment did not alter the acid phosphatase activity of macrophages . However, bactericidal ability of MMS-treated macrophages for unopsonized Escherichia coli was significantly depressed . These results suggest that the avian macrophage is a useful target cell for examining possible relationships between genotoxic and immunotoxic effects of environmental mutagens.

Growth Factors, 1989, 2(1), 31 - 42
Direct demonstration of an autocrine mechanism in EMS-induced, tumorigenic mutants of the growth factor-dependent hemopoietic cell line, FDC-P1; Wilks AF et al.; A growth factor-dependent hemopoietic cell-line, FDC-P1, was treated with a chemical mutagen, EMS, and a number of growth factor-independent variants isolated . Six lines have been extensively analyzed with respect to their growth kinetics, morphology, karyotype, tumorigenicity, and hemopoietic growth factor production . Four lines produced at least one growth factor, subsequently demonstrated to be GM-CSF, while two lines showed no evidence of hemopoietic growth factor production . The observation that the autonomous proliferation of those EMS-derived cell lines that produced GM-CSF can be inhibited by incubation in growth media containing 10-50 microM anti-sense GM-CSF oligonucleotides demonstrated directly that the autonomous behavior of these cells is based on an autocrine mechanism . The induction of the expression of the GM-CSF gene represents a rare class of EMS-induced mutants, and is strongly suggestive of repressor inactivation rather than promoter activation.

Arch Microbiol, 1989, 152(2), 115 - 8
Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12 . Involvement of the glnLG and purR genes in the regulation of codA expression; Andersen L et al.; Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia . Regulation of codA expression was studied by determining the level of cytosine deaminase in E . coli K12 grown in various defined media . Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis . Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides . Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product . Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.

Radiobiologiia, 1989 Jan-Feb, 29(1), 36 - 40
{The effect of pre-irradiation conditions of cultivation on the induction of mutations in E . coli bacteria by gamma radiation}; Tokarova B et al.; The influence of preirradiation cultivation conditions on the survival rate and mutation frequency in gamma-irradiated E . coli AB 1,157 and Hfr has been investigated . Radiosensitivity of cells and mutagenesis have been shown to vary depending on the growth medium . These differences are related to different efficiency of gene expression regulating the inducible SOS-response of cells.

J Neurosci, 1989 Jan, 9(1), 63 - 9
Plasticity of process-bearing glial cell cultures from neonatal rat cerebral cortical tissue; Ingraham CA et al.; The factors and cellular interactions that influence the commitment of cells to specific neural lineages are not well understood . We have used cultured non-neuronal process-bearing (PB) cells from neonatal rat cerebral cortices as a model to assess the influence of various culture conditions on the determination of cells as either astroglia or oligodendroglia . Increasing postseparation plating density was significantly associated (p less than 0.001) with decreasing percentages of glial fibrillary acidic protein (GFAP+) cells, increasing percentages of galactocerebroside (GC+) cells, and increasing percentages of nonstained cells . As the fetal calf serum content of growth medium was increased, the percentage of GFAP+ cells increased, and as the serum content was decreased, the percentage of GC+ cells increased . Evidence of minimal cell proliferation and the observation of PB cells that coexpressed GFAP and GC supported the conclusion that PB cells switched their phenotypic expression from GFAP+ in serum to GC+ in serum-free medium . PB cells exhibited plasticity in their phenotypic expression as cells grown for 9 d in serum-free medium were still responsive to the effects of serum, while cells grown for 6 d in serum were refractory to serum withdrawal . This research has demonstrated the plasticity of PB cells separated from polygonal astroglia as they expressed GFAP in the presence of serum and GC in serum-free medium.

Mol Biochem Parasitol, 1989 Jan 1, 32(1), 25 - 37
De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum; Krungkrai J et al.; Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu) . The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium . The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography . Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.

Cytobios, 1989, 60(242-243), 151 - 5
Denaturing effect of acridine orange and adriamycin; Cerdan ME et al.; Acridine orange and adriamycin have a very low denaturing effect on DNA isolated from Chinese hamster cells but strongly increase heat-induced denaturation . When these intercalating agents are added to the growth media they modify the sensitivity to heat denaturation of DNA extracted from treated cultures when compared with untreated cultures . This effect correlates with a considerable increase in the disruption of chromosomal ultrastructure.

Gig Tr Prof Zabol, 1989, (12), 30 - 4
{The solubility of welding dusts and their cytotoxicity}; Gorban' LN et al.; It was established as a result of cytotoxicity studies of 15 welding dusts of different compositions in experiments on embryonal fibroblast cell cultures that the biologic activity of these substances depends on the solubility in growth media of their major components: alkaline and alkaline earth metals, iron, cremnium and, to a vast degree, that of the manganese . Conclusion has been made that a comparative assessment of welding dusts' biological activity on cell cultures should involve the kinetic parameters of their solubility in growth and supportive media, and the duration of the substances' affecting the cell cultures should be long enough to reveal the combined action of its major ingredients.

Connect Tissue Res, 1989, 21(1-4), 239 - 44; discussion 245-6
Calcium phosphate mineralization; Nancollas GH et al.; Although it is often assumed that the thermodynamically most stable hydroxyapatite is a suitable prototype for biological minerals, it is now generally accepted that other phases such as dicalcium phosphate dihydrate and octacalcium phosphate as well as defect apatites and carbonated apatites may participate . The Constant Composition kinetics method, has been used to show that defect apatites may be formed with non-stoichiometric coefficients that depend upon the pH of the growth medium . Important factors in analyzing these experiments are the initial surface modification and ion exchange processes involving hydrogen and calcium ions following inoculation of the supersaturated solutions . Proteins and other macromolecules which may inhibit the rate of growth of calcium phosphates in supersaturated solutions are able to enhance the nucleation of these phases when immobilized on inert surfaces.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1989, 56(5), 293 - 8
Evidence of an acquired increase in mitogenesis in streptozotocin-diabetic rats, apparently relating to some tissue factor; Druvefors P et al.; We have previously reported that rats which have been suffering from streptozotocin-diabetes for 4 weeks show a supranormal mast cell mediated mitogenesis in mesenteric windows and in the skin; this late emerging, augmented mitogenic responsiveness appears, to be unaffected by insulin per se . To test whether this increased proliferogenic response is effected by some acquired quality within the tissue rather than a systemic factor in the blood, we studied mast cell mediated mitogenesis in organ-cultured intact mesenteric windows from rats with diabetes of 4 weeks' duration, using a biochemically-defined serum-free growth medium . Mast cells were activated by Compound 48/80 and their secretion was quantified biochemically in terms of histamine release . The mast cell-dependent mitogenic reaction in the predominant, morphologically discrete fibroblasts and mesothelial cells was quantified photometrically using Feulgen-absorption analysis of individual cell nuclei, and by determination of the mitotic index . Both types of target cell responded to a significantly greater degree mitogenically in diabetic compared with control tissue . This finding suggests that a considerable part of the increased mitogenic responsiveness previously observed in diabetic animals in vivo is causally related to some tissue-bound, i.e., cellular and/or extracellular factor(s) acquired during the course of the disease.

Int J Radiat Biol, 1989 Jan, 55(1), 15 - 26
Radiation damage to glucose concentrating capacity and cell survival in kidney tubule cells: effects of fractionation; Parkins CS et al.; The glucose concentrating capacity of cultured LLC-PK1 kidney epithelial cells has been measured after single and fractionated doses of X-rays . Steady-state glucose concentrating capacity (ratio of glucose concentration inside to outside cell) can be measured using radiolabelled analogues of glucose which are actively transported but not metabolized . These cells can be stimulated to increase their glucose concentrating capacity (up-regulation) by a reduction in the glucose concentration of the growth medium . However, after X-ray irradiation the cells have a reduced capacity to respond to up-regulation . This effect can be measured 7 days after irradiation and before radiation-induced cell killing affects the cell population . The previously reported radiosensitivity of this function to single doses of X-rays (in the range 1-16 Gy) was confirmed . Surprisingly, no significant sparing of this effect could be measured by fractionation of the X-ray dose into two or four fractions . However, the cells showed a significant fractionation effect if clonogenic survival was measured using the standard cell survival assay . These early effects have different fractionation response from the later phases of tissue damage, measured months to years after irradiation, which do show sparing due to fractionation and are thought to be mainly due to changes in cell survival . The lack of sparing by fractionation to the functional damage may suggest a different target from that which determines cell survival . These results support the hypothesis that radiation damages cellular functions, separately from cell replication.

Int J Radiat Biol, 1989 Jan, 55(1), 105 - 17
Trypsinization and the radiosensitivity of mitotic and log phase Chinese hamster V79 cells exposed to 250 kVp X-rays; Reddy NM et al.; We studied the influence of trypsin-induced morphological changes on the radiosensitivity of cells plated at either low (4-600/cm2) or high (2 x 10(4)/cm2) density and grown overnight before treatments . Trypsin treatment induced contraction and rounding of spread cells . The radiosensitivity of cells trypsinized and plated either: (1) immediately before {(a) D0 = 1.7 Gy for cells at low-, and (b) 1.5 Gy at high-density} or (2) immediately after (D0 = 1.6 Gy, high-density cells) irradiation was higher than that of (3) cells at high density, irradiated and delayed plated {cells remained spread until the completion of potentially lethal damage repair (PLDR) and trypsinization, D0 = 2.2 Gy}, and (4) cells at low density which were neither delayed plated nor trypsinized (i.e . remained spread) after irradiation (Do = 2.4 Gy) . These data show that PLDR is reduced in trypsin-treated cells of both high (compare 1b and 2 with 3) and low (compare 1a with 4) density cultures; the latter comparison provides a direct measure of the trypsin effect . Since the comparison between conditions 1 and 2 vs . 3 and 4 is of round vs . spread cells, PLDR appears to be influenced by the cell's morphological state . Kinetic studies showed that when cells were incubated in growth medium to recover from trypsin-induced effects before irradiation, the radiation sensitivity of spread cells (plated in situ), but not of those remaining rounded (in suspension until plating and irradiation), decreased and became equal to that of delayed plated high-density cells . Neither irradiated cells treated with hypertonic saline, nor mitotic cells, showed the trypsin effect . From these results we suggest that: (1) trypsin-induced cell contraction affects the ability of cells to repair radiation damage, (2) spread cells are better able to repair PLD than rounded cells, (3) immediate plating survival of cells in high-density cultures may not represent their intrinsic radiosensitivity and (4) cell-to-cell contact is not necessary for log phase cells to repair PLD.

Prostate, 1989, 15(4), 315 - 25
Separation of mature rat ventral prostate epithelial and fibroblast cells; Montpetit ML et al.; Several techniques for the separation of rat ventral prostate cells, using density gradient centrifugation or mechanical means have been published, yielding fibroblast and epithelial cell populations of varying purities and viability . These techniques are often tedious, yield relatively limited numbers of cells, demand considerable technical expertise, and result in the isolation of cells of limited viability . Two techniques for the isolation and establishment of epithelial and fibroblast cell cultures from mature rat ventral prostate are described here . Collagenase/trypsin digestion of the tissue yields a single-cell suspension of both cell types that are separated on Percoll isopycnic centrifugation gradients . A continuous gradient system allows for the separation of a greater number of cells with very high degrees of purity . A second technique based on a step-gradient system produces reproducible subfractionation of the epithelial cell component of the prostate within a considerable shorter period of time . An improved medium for plating epithelial and fibroblast cells has also been developed . The separated cells are plated on collagen and/or fibronectin-coated dishes in a serum-free plating medium that is later replaced with a serum containing growth medium . The plating medium greatly increases the plating efficiency of the isolated cell types, particularly the epithelial cells.

Biochem Int, 1989 Jan, 18(1), 197 - 202
Further serum related alterations in the activities of some membrane marker enzymes in normal and virus transformed cells; Tenang EM et al.; The activities of membrane marker enzymes in normal (3T3) and simian virus transformed mouse cells (SV3T3) are affected not only by densities of cultures but also by the sera types used in the growth media . We have assayed the levels of 5'nucleotidase, monoamine oxidase and rotenone insensitive NADH ferricyanide reductase in these cells grown to sparse and confluent cultures in medium supplemented with 10% newborn calf serum (n.c.s.) or in medium supplemented with 10% foetal bovine serum (f.b.s.) . It was found that in 3T3 cells grown in 10% f.b.s . the transition from sparse to confluent cultures was associated with a reduction in the activities of the marker enzymes while in those grown in 10% n.c.s., the activities of these enzymes increased . In the SV3T3 cells, the activities of all the enzymes except for monoamine oxidase decreased from sparse to confluent culture densities in cells grown in 10% n.c.s . whereas in those grown in 10% f.b.s . there were no significant change in the activities of the enzymes over the same culture densities . The results suggest that the marker enzymes are affected by sera types and culture densities.

Exp Eye Res, 1989 Jan, 48(1), 99 - 106
Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose; Li WY et al.; Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined . When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively . In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose . When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase . Phosphatidylinositol bisphosphate (PIP2) . Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol . The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium . However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation . These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions . Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.

J Rehabil Res Dev, 1989 Winter, 26(1), 1 - 14
Artificial nerve graft compared to autograft in a rat model; Rosen JM et al.; A study was made to compare the regeneration of rat peroneal nerve across a 0.5 cm gap repaired with a sutured autograft (SAG) versus an artificial nerve graft (ANG) . The ANG model is composed of a synthetic biodegradable passive conduit made of polyglycolic acid (PGA) and a synthetic growth medium composed of hypoallergenic collagen . Axonal regeneration in short-term animals (1 and 4 months) was evaluated by qualitative histology only, while in long-term animals (17 to 21 months) quantitative histology and electro-physiology were used in addition to qualitative histology . This study reveals that axons do regenerate through this ANG model, but electrophysiological analyses show that the axonal regeneration is statistically inferior to that in the SAG . There was no significant statistical difference in the quantitative histological data.

Carcinogenesis, 1989 Jan, 10(1), 123 - 30
The production of chromosomal alterations in human lymphocytes by drugs known to interfere with the activity of DNA topoisomerase II . I . m-AMSA; Andersson HC et al.; The frequencies of chromosomal aberrations and sister chromatid exchanges (SCE) were studied in human peripheral lymphocytes after exposure of whole blood cultures to the antitumour agent m-AMSA . Chromosome-type aberrations were found in cells exposed before stimulation or during the G1-phase of the cell cycle . Chromatid-type aberrations were obtained in cells exposed during the S- and G2-phases, but also in cells exposed the G1-phase or before stimulation . Treatment of lymphocytes with m-AMSA in the G1-phase or before stimulation had the additional effect of strongly increasing the frequency of SCE . When unstimulated lymphocytes were exposed to m-AMSA, the frequencies of SCE and chromatid-type aberrations, but not the frequency of chromosome-type aberrations, could be strongly reduced by holding the cells in F-10 for 2-4 h before stimulation, or by changing the growth medium after stimulation . A 1-h incubation in growth medium was sufficient for obtaining this reduction . The medium in which the m-AMSA-treated cells were incubated for the first 3 h after stimulation proved to be capable of inducing chromatid-type aberrations in late S/G2-phase and SCEs in the S-phase . These observations show that the chromatid-type aberrations and SCEs obtained by exposing unstimulated lymphocytes to m-AMSA were not produced during the treatment itself, but after stimulation at an advanced stage of the cell cycle by an active substance (m-AMSA or a metabolite of m-AMSA) released into the medium during the first hours of incubation . Post-treatments in G2 with 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea strongly enhanced the frequency of chromatid-type aberrations obtained after treatment of stimulated or unstimulated lymphocytes with m-AMSA . Post-treatments in unstimulated or G1-phase lymphocytes with ara-C did not influence the frequency of chromosome-type aberrations induced by m-AMSA at these stages, but strongly enhanced those induced by X-rays.

Folia Microbiol (Praha), 1989, 34(3), 185 - 94
Production of acid and alkaline phosphatases by Myxococcus coralloides; Gonzalez F et al.; Acid and alkaline phosphatase of Myxococcus coralloides were examined during vegetative growth in a liquid medium . Two extracellular phosphatases and two cell-bound phosphatases, acid and alkaline in both cases, were produced . The phosphatase production was unaltered by the presence of high concentrations of inorganic phosphate . Both enzymes were produced constitutively . These two hydrolases were released into the growth medium during the exponential growth phase (approximately 10% of total activity) . The production of these enzymes was modified by the presence of organic acids and metal ions in the medium.

Nephrol Dial Transplant, 1989, 4(1), 1 - 8
Autoantibodies to glomerular antigens in patients with Wegener's granulomatosis; Abbott F et al.; Patients with Wegener's granulomatosis have autoantibodies to a neutrophil cytoplasmic antigen . As these patients often present with severe glomerulonephritis we investigated whether the same autoantigen was expressed in glomeruli by using both cultured human glomerular cells and frozen normal human kidney sections . Glomeruli were isolated from normal human kidney cortex by differential sieving and plated on to coverslips in growth medium containing various supplements . Cell types were identified by a series of markers and designated either epithelial, endothelial or mesangial . In single and double staining experiments, glomerular cells were incubated with a monoclonal antibody to the neutrophil cytoplasmic antigen (MCAW8), control mouse monoclonal IgG, sera from patients with Wegener's granulomatosis and control normal human serum . MCAW8 and Wegener's patient sera bound specifically to cultured epithelial and endothelial cells . MCAW8 was also found to bind to the glomerular epithelium in frozen sections . We conclude that autoantibodies to glomerular antigens are present in serum of patients with Wegener's granulomatosis and may be important in the pathogenesis of rapidly progressive glomerulonephritis.

Biol Met, 1989, 2(3), 168 - 73
Silver accumulation in Pseudomonas stutzeri AG259; Gadd GM et al.; Silver toxicity to Pseudomonas stutzeri AG259 was strongly dependent on the NaCl concentration in the medium, which reduced the availability of Ag+ by precipitation as AgCl . Accumulation of Ag by growing cultures was low being less than or equal to 7.5 nmol (mg dry mass)-1 over all treatments examined . The presence of NaCl in the growth medium did not markedly affect the amounts of Ag accumulated by the cells but influenced toxicity as manifest by a lag period which was greatest at low NaCl concentrations (less than or equal to 0.1% mass/vol.) . In NaCl-free medium, P . stutzeri did not grow in the presence of 0.5 mM AgNO3 in contrast to Ag-free controls . The majority of Ag accumulation by resting cells of P . stutzeri occurred within 1 min of incubation and there was little difference in uptake capacities between cells previously grown in the absence or presence of AgNO3 . Lowest amounts of Ag uptake by resting cells occurred when suspended in 1 mM Mes pH 6.5, containing 1% (mass/vol.) NaCl . Prior exposure of P . stutzeri to Cu(NO3)2 resulted in a marked reduction in Ag uptake when suspended in 1 mM Mes pH 6.5, containing 0.5 mM AgNO3.

Perit Dial Int, 1989, 9(4), 341 - 7
Isolation and propagation in vitro of peritoneal mesothelial cells; Hjelle JT et al.; Mesothelial cells lining the peritoneal cavity are the primary site of molecular exchange during peritoneal dialysis, a life support system for over 50,000 patients worldwide . In this study, techniques are described for the isolation and propagation in culture of peritoneal mesothelial cells from rats and rabbits . For comparison, mesothelial cells were also obtained from the serosal surface of human colonic tissue . By electron microscopy the cultured cells were found to exhibit microvilli, a well-developed endoplasmic reticulum and golgi apparatus, micropinocytotic vesicles, and lipid-filled intracellular vesicles . Immunochemical probes revealed the expression by these cells in vitro of cytokeratin, fibronectin, vimentin, and keratin, but not von Willebrand factor . Mesothelial cells from rat, rabbit, and human exhibited contact inhibition, but differences in growth rates and dependence on supplements to the growth media . This work provides a multispecies comparison of the behavior of mesothelial cells in vitro for the purpose of developing an experimental system for the study of mesothelial cell biology and the role of these cells in peritoneal dialysis.

Lab Delo, 1989, (7), 68 - 70
{A coagglutination method in the identification of Bordetella genus microbes}; Bakulina NA et al.; Trials of the coagglutination test for the identification of Bordetella bacteria and for the detection of B . pertussis in the material from healthy and sick children have demonstrated the possibility of using this test for the detection of B . pertussis in mixed cultures on the third day after preliminary inoculation in the growth medium (casein-coal agar) and as a test for the differentiation between B . pertussis and gram-negative bacteria similar to it by their morphologic and serologic characteristics.

Avian Dis, 1989 Jan-Mar, 33(1), 140 - 9
Further studies of Mycoplasma gallisepticum serum plate agglutination antigen grown in medium with artificial liposomes substituting for serum; Ahmad I et al.; Frey's medium supplemented with artificial liposomes substituting for serum was evaluated for Mycoplasma gallisepticum (MG) serum plate agglutination (SPA) antigen . Antigens prepared in batch (static) culture were compared with antigens grown in a fermenter . All batch-grown MG liposome antigens were highly sensitive, specific, and resulted in a greater yield compared with fermenter-grown liposome antigens . Compared with antigens prepared in Frey's medium with 12% swine serum (regular FMS) or with commercial SPA antigens, liposome antigens had a higher degree of specificity; however, they were similar in sensitivity and antigen yield . The only growth parameter to affect the yield per liter of batch-grown liposome antigen was the concentration of liposomes in the growth medium . The reduced yield and sensitivity of antigens grown in a fermenter may have been due to autoclaving the medium instead of sterilizing by filtration . There was no obvious difference between patterns of serum-medium-grown, liposome-medium-grown, or commercial SPA antigens upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cancer Res, 1988 Dec 15, 48(24 Pt 1), 7219 - 25
Effects of retinoic acid on morphological features and biological markers of a neoplastic human salivary intercalated duct cell line in culture; Azuma M et al.; Retinoic acid has marked effects on the growth, morphological features, and biological markers of a neoplastic human salivary intercalated duct cell clone in culture, whereas the cell clone was not affected by other retinoids such as retinol and retinal . A cell clone with ultrastructure and biological markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of retinoic acid . Major alterations, such as expression of tonofilaments, Mr 68,000 cytokeratin, and involucrin, were observed in those cells with a phenotype similar to that of keratinizing squamous cells . In addition, the coexpression of Mr 68,000 cytokeratin and carcinoembryonic antigen in these altered cells was found . Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of retinoic acid . After the removal of retinoic acid from the culture, the treated cells returned rapidly to the phenotype of the untreated cells . These findings indicate that reversible differentiation into the keratinizing squamous cells of a neoplastic human salivary intercalated duct cell clone occurs in growth medium containing retinoic acid.

J Biol Chem, 1988 Dec 5, 263(34), 18118 - 22
Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae; Perlin DS et al.; The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined . It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells . However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells . The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from {14C}tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants . The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium . These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.

FEBS Lett, 1988 Dec 5, 241(1-2), 15 - 8
The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line; Doostdar H et al.; When grown in the standard Dulbecco's medium the human liver derived Hep G2 hepatoma cell line shows only 10-20% of the cytochrome P-450-dependent mixed function oxidase (MFO) activity of freshly isolated human adult hepatocytes . However, the MFO activities and, to a lesser extent, the activities of UDP-glucuronyltransferase and glutathione-S-transferase can be increased by altering the composition of the growth medium . Modified Earle's medium was more effective in this respect than Williams' E medium and increased the O-dealkylations of ethoxyresorufin, benzyloxyresorufin and pentoxyresorufin 50-, 30- and 10-fold, respectively.

J Biol Chem, 1988 Dec 5, 263(34), 18078 - 85
Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol . Inositol is an inhibitor of phosphatidylserine synthase activity; Kelley MJ et al.; The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis . The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine . Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree . Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol . Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol . The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM) . The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM) . Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis . Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity . Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.

Neurochem Res, 1988 Dec, 13(12), 1163 - 7
Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex; Tholey G et al.; Mg2+, Ca2+, Mn2+, Zn2+, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy . The intracellular volume of cultured neurons was determined to be 2.73 microliters/mg . Intracellular Mn2+, Fe2+, Zn2+, and Cu2+ in the cultivated neurons were 100-200 times the concentrations in the growth medium: Mg2+ and Ca2+ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium . Mg2+, Fe2+, Cu2+ and Zn2+ concentrations in neurons were in the range of ca . 300-600 microM, approximately 2-3 times the values previously reported in glial cells; Ca2+ and Mn2+ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells . In neurons, the subcellular distribution of Fe2+, Cu2+, and Mn2+ follows the rank order: cytosol greater than microsomes greater than mitochondria; for Zn2+ the distribution differs as following: cytosol greater than mitochondria greater than microsomes . Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn2+ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells . Enrichment of the culture medium with Mn2+ to 2.5 microM enhanced the Mn-SOD by approximately 33% but the Cu2+-Zn2+ enzyme activity was not modified . The high Mn2+ content, the capacity to accumulate Mn2+, and the predominancy of the Mn-SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.

Appl Environ Microbiol, 1988 Dec, 54(12), 3086 - 91
Laccase-mediated detoxification of phenolic compounds; Bollag JM et al.; The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined . The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol . A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase . The medium was then inoculated with R . praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis . The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol . Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid . The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions . The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.

Appl Environ Microbiol, 1988 Dec, 54(12), 2953 - 8
Metabolism of glyphosate in Pseudomonas sp . strain LBr; Jacob GS et al.; Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp . strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium . Pseudomonas sp . strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date . The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium . Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism . Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM {2-13C,15N}glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp . strain PG2982 . Thus, Pseudomonas sp . strain LBr appears to possess two distinct routes for glyphosate detoxification.

J Cell Biol, 1988 Dec, 107(6 Pt 1), 2097 - 107
Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum; Richardson JM et al.; Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein . The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes . Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with {35S}methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases . Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase . Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors . However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes . In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes . The accumulated intermediate forms of the enzymes, however, were localized to lysosomes . Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes . The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes . Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.

Mol Cell Biol, 1988 Dec, 8(12), 5132 - 9
SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes; Thomas D et al.; In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase . In a previous study we cloned and sequenced the SAM1 gene (D . Thomas and Y . Surdin-Kerjan, J . Biol . Chem . 262:16704-16709, 1987) . In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption . It has been sequenced and compared with the SAM1 gene . The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes . Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions . The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions . The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains . This raises the question of how the two AdoMet synthetases in S . cerevisiae interact to control AdoMet synthesis.

Mol Gen Genet, 1988 Dec, 215(1), 19 - 25
Secretion and export of IGF-1 in Escherichia coli strain JM101; Obukowicz MG et al.; The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101 . Several strain or plasmid modifications were tried to increase export of periplasmic (processed) IGF-1 into the growth medium of JM101 . These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E . coli or yeast optimized IGF-1 codons . None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101 . Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble . This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium . Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.

Genetics, 1988 Dec, 120(4), 909 - 22
Saccharomyces cerevisiae cho2 mutants are deficient in phospholipid methylation and cross-pathway regulation of inositol synthesis; Summers EF et al.; Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques . Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase . Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE . In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME) . However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability . The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis . Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase . This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium . We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo . The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.

J Cell Biol, 1988 Dec, 107(6 Pt 1), 2389 - 99
Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells; Siliciano JD et al.; Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B . R., J . D . Siliciano, M . S . Mooseker, and D . A . Goodenough . 1986 . J . Cell Biol . 103:755-766) . In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1 . In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex . If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm . After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis . However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized . ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium . These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane . An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.

Arch Biochem Biophys, 1988 Dec, 267(2), 479 - 89
Polyamine transport in Neurospora crassa; Davis RH et al.; Polyamine transport in Neurospora crassa is concentrative and energy dependent in a dilute buffer . The saturable systems governing the uptake of putrescine (Km = 0.6 mM), spermidine (Km = ca . 0.24 mM), and spermine (Km = 0.07 mM) share components, as indicated by mutual inhibition among the polyamines . In addition, nonsaturable components prevail for putrescine and spermidine, particularly the former . Radiolabeled substrates, once in the cell, are released only slowly, even if unlabeled polyamines are included in the incubation medium . Permeabilization of cells with n-butanol leads to partial release of internalized 14C-polyamines, and the remainder is almost wholly exchangeable with added, unlabeled polyamine . Polyamine uptake was inhibited by the polyamines themselves and by a polyamine analog, methylglyoxal bisguanylhydrazone, but only weakly and incompletely by the basic amino acids arginine and ornithine . Uptake of putrescine and spermidine was inhibited by monovalent cations, Ca2+, and certain other components of the growth medium . As a result, uptake from the growth medium was very slow and largely by way of the nonsaturable uptake mechanism.

Carcinogenesis, 1988 Dec, 9(12), 2147 - 54
Inhibitors of ADP-ribosyl transferase suppress the mitogenic actions exerted by tumour promoters, but not those evoked by peptide mitogens, in primary neonatal rat hepatocytes; Romano F et al.; A significant stimulation of the 24-h (between day 4 and 5 in vitro) new DNA synthetic activity was elicited in primary neonatal rat hepatocytes kept in low-calcium (0.01 mmol/l) HiWoBa2000 synthetic medium by the addition of a single dose (10(-10) mol/l) of each of several tumour promoters {i.e . 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital, nafenopin, saccharin, teleocidin, benzoyl peroxide butylhydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), lindane, clofibrate and melittin} . Even hormones {e.g . epidermal growth factor (EGF), glucagon and insulin at 10(-10) mol/l} and EGF-like acting drugs (i.e . imidazole and indomethacin, at 10(-11) mol/l) similarly enhanced with respect to untreated controls the 24-h flow into S phase of the primary hepatocytes on condition, however, that the cells were incubated in a high- (i.e . 1.8 mmol/l) and not a low-calcium HiWoBa2000 medium . Xenobiotics, peptide mitogens and EGF-like acting drugs also enhanced the in vitro hepatocellular mitotic activity . The growth-stimulatory effects of the aforementioned eleven tumour promoters were entirely suppressed by the simultaneous addition to the growth medium of a fully effective dose (10(-4) - 10(-3) mol/l) of agents, such as 3-aminobenzamide (3-ABA), 3-methoxybenzamide (3-MBA) or nicotinamide (NA), that are known to inhibit the activity of ADP-ribosyl transferase (ADPRT) . However, under the same conditions these inhibitors hampered neither the basal DNA synthetic and mitotic activities of spontaneously cycling hepatocytes nor the stimulation of the hepatocellular growth processes evoked by peptide mitogens and EGF-like acting drugs . Quantitative autoradiographic investigations showed that the incorporation of the ADP-ribose precursor and ADPRT substrate {3H}NAD into nuclear macromolecules of gently digitonin-permeabilized hepatocytes was negligible in the untreated cultures, whereas it was strikingly and nearly steadily increased by a 2-, 8- and 24-h exposure to a fully mitogenic dose (10(-10) mol/l) of TPA, thereby revealing that an early, significant and roughly steady activation of the nuclear ADPRT had taken place in the phorbol ester-treated liver parenchymal cells . The simultaneous addition of 3-ABA (10(-4) mol/l) not only fully checked the mitogenic effects of TPA, but even suppressed about two-thirds of the TPA-elicited nuclear incorporation of {3H}NAD by the permeabilized hepatocytes, thus showing that a significant curtailment of the TPA-activated ADPRT did occur is association with the abatement of the mitogenic effects of TPA by this inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

J Virol Methods, 1988 Dec, 22(2-3), 319 - 28
The effect of dexamethasone on the detection of cytomegalovirus in tissue culture and by immunofluorescence; Thiele GM et al.; With the development of both monoclonal antibodies to the immediate early nuclear antigen (EA) of cytomegalovirus (CMV) and different methods used for its detection, the time required for diagnosis of infection has become significantly shorter . However, discrepancies between tissue culture (TC) and the EA assay methods have raised questions concerning which method is more sensitive and whether a positive test result truly represents infection or latency . We have found that dexamethasone (Dex), when incorporated into the growth medium at a concentration of 10(-5) M, decreases the variability between the two techniques and increases the sensitivities of both TC and EA assay . However, for Dex to be effective, it was necessary to prepare, seed and grow the indicator cells in the presence of 10(-5) M Dex . Additionally, the cells had to be in contact with Dex for approximately 24 h before any effect was observed . Except for this step, all other procedures were the same as have been described elsewhere . In this study, four methods were compared: TC, TC with Dex (TC-D), EA, and EA with Dex (EA-D) . Of 251 clinical specimens (200 microliter/well), 46 (18%) were positive for CMV . Of the 46, 30 (65%) were positive by TC, 39 (85%) by TC-D, 39 (85%) by EA, and 42 (91%) by EA-D . Without Dex the combination of TC plus EA detected only 41 of 46 (84%) positive samples . In contrast, the presence of Dex allowed detection of all 46 positive samples by TC-D and EA-D . Also, more fluorescent forming units (FFU) were detected by EA-D than EA (mean 14.8 FFU), and cytopathic effect was detected sooner (mean 9 days) by TC-D than by TC . Dex significantly increased the detection of CMV by TC (P less than 0.05) . In addition, the use of any one of these methods alone did not effectively detect all positive samples . We suggest the combination of TC-D and EA-D for detection of all samples positive for CMV and to detect other viruses that may be missed by the EA method.

Gene, 1988 Nov 30, 71(2), 339 - 48
A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis; MacRae WD et al.; The cloning and sequencing of an Aspergillus niger gene encoding a secreted form of phosphate-repressible acid phosphatase by complementation of a pacA (phosphate-repressible acid phosphatase) mutant of Aspergillus nidulans is described . The gene contains two introns, 201 and 265 nt in length, and codes for a 1.6-kb transcript . Both phosphate concentration and pH of the growth medium affect the level of expression of the gene in A . niger . Similar regulation is observed in A . nidulans transformants . A putative signal peptide, resembling known signal sequences of yeast, is identified.

Gene, 1988 Nov 30, 71(2), 233 - 46
Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation; Riba L et al.; Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia . The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported {Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199} . In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources . Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium . We have identified a single and apparently invariant transcript for several metabolic growth conditions . We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions . Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.

J Biol Chem, 1988 Nov 5, 263(31), 15845 - 8
Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor; Lucore CL et al.; To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells . Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells . Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases) . Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h . Factors responsible were found to be trypsin-sensitive and dialysis-resistant . Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response . EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner . Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h . The response was not attenuated by cycloheximide (25 micrograms/ml) . Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase . Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold) . We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.

Cancer Res, 1988 Nov 1, 48(21), 6033 - 6
Reduction of epidermal growth factor binding in human breast cancer cell lines by an alkyl-lysophospholipid; Kosano H et al.; The effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl lysophospholipid derivative, on the binding of epidermal growth factor (EGF) to human breast cancer cell lines (MCF-7, ZR-75-1, and BT-20), the human epidermoid cancer cell line (A431), and the rat fibroblast cell line (NIH3T3) were investigated . The addition of 10 micrograms/ml ET-18-OCH3 to the growth medium reduced the binding of EGF to hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) and A431 but did not change that to the hormone-independent breast cancer cell line (BT-20) . ET-18-OCH3 suppressed the EGF-binding prior to the onset of its inhibitory action on cell growth in MCF-7 and ZR-75-1 . Scatchard plot analysis demonstrated that ET-18-OCH3 reduced the number of EGF receptor sites without affecting the affinity of EGF receptors in MCF-7 and ZR-75-1 . Both EGF-binding and cell growth in NIH3T3 were not changed by treatment with 10 micrograms/ml ET-18-OCH3 . These results suggest that ET-18-OCH3 inhibits the growth of hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) by reducing the binding capacity of EGF receptors and consequently by disturbing the transfer of a variety of growth-promoting signals.

Anal Biochem, 1988 Nov 1, 174(2), 415 - 22
A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy; Hutson TB et al.; A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h . The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors . Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed . Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components . Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence . Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h . Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages . Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium . ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis . Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.

J Protozool, 1988 Nov, 35(4), 541 - 6
Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1; Larsen J et al.; The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose . The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e . from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium . The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle . The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle . The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle . Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.

J Bacteriol, 1988 Nov, 170(11), 5208 - 15
lac fusion analysis of the bet genes of Escherichia coli: regulation by osmolarity, temperature, oxygen, choline, and glycine betaine; Eshoo MW; The synthesis of glycine betaine, a powerful osmoprotectant, from its precursor, choline, is a function of the bet genes . The bet genes code for the high-affinity transport of choline and the enzymes for its conversion to glycine betaine . These genes map at 7.5 min on the E . coli chromosome and are contained on the conjugative plasmid F'2 . To study the transcriptional regulation of the bet genes in response to various environmental conditions, a collection of 30 lac operon fusions was isolated by utilizing the bet genes contained on F'2 . Four osmoregulated bet loci (betA, betB, betC, and betT) were identified based on biochemical, regulatory, and merodiploid analysis of these fusions . All of the bet fusions demonstrated a 7- to 10-fold increase in transcription in response to increases in the osmotic strength of the growth medium . Choline further induced expression of lac fusions at the betA, betB, and betT loci when the cells were grown under conditions of osmotic stress . The end product of the pathway, glycine betaine, was a corepressor of choline induction for fusions at the betA and betT loci . Expression of the betA, betB, and betT loci was reduced 7- to 10-fold under anaerobic conditions . In addition, expression of the betB and betT loci was reduced when the cells were grown in high osmolarity at 16 degrees C . These studies demonstrate that the expression of the bet genes is under the control of several environmental stimuli.

Arch Biochem Biophys, 1988 Nov 1, 266(2), 470 - 7
Control of bacteriochlorophyll accumulation by light in an aerobic photosynthetic bacterium, Erythrobacter sp . OCh 114; Shioi Y et al.; The effect of light on bacteriochlorophyll (Bchl) accumulation as well as the activity of two enzymes in the initial step of the tetrapyrrole biosynthetic pathway was examined in an aerobic photosynthetic bacterium, Erythrobacter sp . strain OCh 114 . Light clearly regulated the Bchl and carotenoid accumulation, completely suppressing their levels at high light intensity . However, porphyrin and Bchl precursors were not found in either the cells or the growth medium of lighted culture . The level of Bchl showed an inverse relationship to the light energy flux . Kinetic studies showed a Hill coefficient of n = 3.3 (r = 0.973), indicating a positive cooperativity . Bchl accumulation was stopped immediately upon illumination without any lag or overshoot . Despite low Bchl content, the activities of 5-aminolevulinic acid synthetase and porphobilinogen synthase were rather stimulated, but not suppressed by light . The high activity of enzymes coincided with the results that heme contents, particularly cytochrome c and catalase activity, were increased in light-grown cells . These results suggest that light regulated Bchl accumulation, but not Bchl biosynthesis and that the effect of light is to render newly formed pigment molecules unstable.

J Bacteriol, 1988 Nov, 170(11), 5330 - 6
Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12; Sawers G et al.; The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated . Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium . Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression . Complete anaerobic induction required the presence of a functional fnr gene product . However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant . These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions . It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.

J Bacteriol, 1988 Nov, 170(11), 5216 - 23
Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins; Rao NN et al.; We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium . A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium . Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate . A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization . Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth . To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy . The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium . When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did . The degradation of polyphosphate by intact cells of E . coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.

Arch Biochem Biophys, 1988 Nov 1, 266(2), 478 - 85
Inhibition of porphyrin biosynthesis by exogenous 5-aminolevulinic acid in an aerobic photosynthetic bacterium, Erythrobacter sp . OCh 114; Shioi Y et al.; Exogenously administered 5-aminolevulinic acid (ALA) inhibited the formation of bacteriochlorophyll a (Bchl a) in a dose-dependent manner in the aerobic photosynthetic bacterium, Erythrobacter sp . strain OCh 114, under dark growth conditions . The ALA concentration required for half-inhibition after 24-h growth was estimated to be about 3.0 mM . Porphyrin and Bchl precursors were not found in either the cells or the growth medium . The same inhibition was also observed with cytochrome c formation . When ALA was incubated with intact cells, a large amount of ALA was converted to an unknown metabolite . The pH optimum of the conversion was 7.8 . The metabolite did not react with Ehrlich's reagent, but did so with ninhydrin, giving a yellow color . Based on analyses by several techniques including mass spectrometry, ir spectrometry, and paper electrophoresis, it was identified as 4-hydroxy-5-aminovaleric acid (HAVA) . Authentic HAVA prepared from ALA was a competitive inhibitor of the enzyme, porphobilinogen synthase of Erythrobacter . The Ki value for authentic HAVA was calculated to be 2.4 mM from a Dixon plot and the HAVA concentration required for half-inhibition was 17 mM . It is concluded that in Erythrobacter cells, exogenous ALA is converted to the metabolite, HAVA, which is responsible for the inhibition of porphobilinogen synthase as well as that of Bchl a and cytochrome formation.

Biochim Biophys Acta, 1988 Oct 13, 967(1), 49 - 55
Identity of soluble thiamin-binding protein with thiamin-repressible acid phosphatase in Saccharomyces cerevisiae; Nosaka K et al.; Two secretory glycoproteins of Saccharomyces cerevisiae, a soluble thiamin-binding protein and a thiamin-repressible acid phosphatase, were shown to be repressed to a similar extent by excess thiamin in the growth medium . Thiamin-repressible acid phosphatase was co-purified throughout the purification of the soluble thiamin-binding protein . Purified and deglycosylated soluble thiamin-binding proteins exhibited both thiamin-binding and acid phosphatase activity on non-denaturing polyacrylamide gel electrophoresis . Heat treatment of the purified soluble thiamin-binding protein caused a decrease in both activities with a similar inactivation profile . Furthermore, two thiamin-repressible acid phosphatase-defective mutants isolated had no and decreased soluble thiamin-binding activity, respectively . From the results, it was concluded that the soluble thiamin-binding protein is identical to the thiamin-repressible acid phosphatase in S . cerevisiae.

FEBS Lett, 1988 Oct 10, 238(2), 375 - 8
The herbicidally active experimental compound Hoe 704 is a potent inhibitor of the enzyme acetolactate reductoisomerase; Schulz A et al.; Growth inhibition of plants and bacteria by the experimental herbicide Hoe 704 (2-methylphosphinoyl-2-hydroxyacetic acid) was alleviated by the addition of the branched-chain amino acids to growth media . Hoe 704 caused a massive accumulation of acetoin and acetolactate, indicating its direct interference with the branched-chain amino acid biosynthetic pathway . The second enzyme of this pathway, acetolactate reductoisomerase (EC 1.1.1.86), was found to be subject to strong inhibition by Hoe 704 . The inhibition was time-dependent and competitive with the enzyme's substrate, acetolactate . This report establishes acetolactate reductoisomerase as a new target for a herbicidal compound.

Infect Immun, 1988 Oct, 56(10), 2570 - 5
Regulation of hemolysin expression in Actinobacillus pleuropneumoniae serotype 1 by Ca2+; Frey J et al.; Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, secretes a hemolytic activity which is thought to be a factor involved in the pathogenesis of the disease . The biosynthesis of hemolysin by serotype 1 strain 4074 was strongly dependent on the activity of free Ca2+ in the growth medium . At activities of free Ca2+ below 50 microM, very low hemolytic activities could be detected in the growth medium and in cell extracts . Maximal hemolytic activities of up to 400 hemolytic units per ml could be measured in growth medium containing free Ca2+ activities above 3 mM . Other bivalent cations did not stimulate the production of hemolysin . Neither the growth rate nor the secretion of hemolysin was affected by increasing Ca2+ concentrations in the medium . The hemolysin of serotype 1 did not require Ca2+ as a cofactor for the lysis of erythrocytes . Ca2+ induced the expression of a 105-kilodalton protein, which was secreted . This protein comigrated with purified hemolysin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited hemolysin activity upon purification . Inhibition experiments with rifampin suggest that the hemolysin of A . pleuropneumoniae is regulated by Ca2+ at the transcriptional level . The threshold of hemolysin induction was around 700 microM free Ca2+, a concentration which is similar to that found in blood serum . The Ca2+-inducible hemolysin represents a novel type of positively regulated bacterial gene expression.

Dev Biol, 1988 Oct, 129(2), 476 - 94
Cell cycle withdrawal without concomitant differentiation: analysis of a G1-specific temperature-sensitive murine myoblast cell line; Compton RS et al.; Skeletal muscle differentiation is accompanied by the withdrawal of the proliferating myoblasts from the cell cycle in the G1 phase . We showed earlier that the length of G1 and the timing of the differentiative transition could be controlled in large part by the composition of the culture medium . In this study we have asked whether a G1 arrest imposed independently of the culture medium is sufficient to elicit the differentiative response . To examine this possibility we have characterized a new G1-specific ts murine myoblast line . This line, ts-36, was identified as a G1-specific mutant on the basis of four criteria: prolonged viability at the nonpermissive temperature (npt), the kinetics of cell cycle withdrawal and reentry in temperature shift experiments, the ability of the cells to differentiate at the npt in low-growth medium, and, finally, the observation that, by the criterion of flow microfluorometry, the mutant cells block at the G1 landmark in the cell cycle . A ts-imposed G1 arrest of up to 96-hr duration is by itself insufficient to activate the differentiative program in ts-36 cells cultured in complete growth medium . The differentiated phenotype is expressed, however, in temperature-arrested cells cultured either in low-growth (conditioned) medium or in a medium from which mitogens have been removed by ultrafiltration . Differentiation can be reversed by refeeding with complete growth medium . The effects of growth medium can be mimicked by FGF to the extent of inhibiting activation of the differentiative program in temperature-arrested ts-36 cells and in eliciting downregulation of muscle-specific contractile protein synthesis . Extrapolating from these observations suggests that growth factors may have more than one role in myogenesis in vitro . They not only stimulate proliferation, but also inhibit differentiation in the absence of proliferation . Examining the kinetics of withdrawal from the cell cycle indicates that ts-36, cultured in conditioned medium blocks at the npt restriction point rather than the conditioned medium block . Our results suggest that two conditions must be met to trigger myogenic differentiation in vitro . Withdrawal from the cell cycle in G1 alone is not sufficient . Reduction of the mitogen level in the medium below a threshold level is an obligate condition for phenotypic expression.

Tsitologiia, 1988 Oct, 30(10), 1273 - 6
{Recovery of the viability of Chinese hamster V79-4 cells after combined exposure to hyperthermia and radiation}; Nasonova EA et al.; The survival of cells overheated (42 degrees C) before gamma irradiation is increased by holding them in the growth medium at 37 degrees C before treatment with hypertonic NaCl solution . The substantial synergistic effect of hyperthermia and radiation takes place when the cells are treated with a 1.5 M NaCl solution immediately after the combined action of these inactivating factors . The synergistic effect is decreased by holding the cells in the nutrient medium at 37 degrees C for 4 hours before hypertonic treatment.

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