Growth media

Biochem J, 1991 Dec 1, 280 ( Pt 2), 545 - 8
The plasma membrane ferrireductase activity of Saccharomyces cerevisiae is partially controlled by cyclic AMP; Lesuisse E et al.; The plasma-membrane-bound ferrireductase activity of ras1 and ras2 mutants of Saccharomyces cerevisiae is not induced in response to iron limitation . This phenotype was suppressed by the bcy1 mutation in ras2 but not in ras1 mutants . The cellular haem content of ras-1-bearing strains decreased dramatically when cells were grown in semi-synthetic medium (low yeast extract content), which could account for their very low ferrireductase activity . The ferrireductase activity of cdc25 and cdc35 mutants dropped when the cells were shifted to a non-permissive temperature . This drop was prevented in the double mutant cdc35 sra5 by adding cyclic AMP to the growth medium . We propose that ferrireductase activity is under the control of a cyclic AMP-dependent protein phosphorylation.

J Mol Biol, 1991 Nov 20, 222(2), 281 - 300
Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity . Implications for protein-DNA interactions in vivo; Cayley S et al.; The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium . The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM . Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity . The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight . Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity . The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule . Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases . Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb . K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity . The amount of K+ greatly exceeds that of Glu- . Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity . As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution . In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells . The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions . Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)

Microb Pathog, 1991 Nov, 11(5), 317 - 23
Iron represses the expression of CFA/I fimbriae of enterotoxigenic E . coli; Karjalainen TK et al.; Experiments were designed to study the effect of iron on the expression of CFA/I fimbriae by enterotoxigenic E . coli (ETEC) . Addition of 0.05 mM ferrous sulfate to growth media decreased CFA/I antigen and fimbrial production by the CFA/I-positive ETEC strain H-10407 as measured by quantitative ELISA and hemagglutination assay . The repressive effect was reversed by the addition of the iron chelators, sodium citrate or dipyridyl . With a CFA/I subunit gene promoter-lacZ fusion, it was found that the activity of the subunit gene promoter was significantly higher in the presence of iron chelators than in medium containing iron in the fur+ strain DHB24 . This difference was not observed in the fur mutant strain SBC24, suggesting that the global E . coli metalloregulatory protein Fur (ferric uptake regulation) is involved in the repression . The repressor may bind to the promoter of the CFA/I subunit gene since several potential Fur-binding sites were identified in the promoter area.

Biochim Biophys Acta, 1991 Nov 11, 1090(3), 326 - 32
Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae; Gaynor PM et al.; Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae . Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S . cerevisiae cells supplemented with phospholipid precursors . The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells . The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol . Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively . These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline . CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively . However, there was no regulation in response to inositol when the mutants were not supplemented with choline . This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway . The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively . CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants . Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5991 - 7
Manipulation of the 'zinc cluster' region of transcriptional activator LEU3 by site-directed mutagenesis; Bai YL et al.; The transcriptional activator LEU3 of Saccharomyces cerevisiae belongs to a family of lower eukaryotic DNA binding proteins with a well-conserved DNA binding motif known as the Zn(II)2Cys6 binuclear cluster . We have constructed mutations in LEU3 that affect either one of the conserved cysteines (Cys47) or one of several amino acids located within a variable subregion of the DNA binding motif . LEU3 proteins with a mutation at Cys47 were very poor activators which could not be rescued by supplying Zn(II) to the growth medium . Mutations within the variable subregion were generally well-tolerated . Only two of seven mutations in this region generated poor activators, and both could be reactivated by Zn(II) supplements . Three of the other five mutations gave rise to activators that were better than wild type . One of these, His50Cys, exhibited a 1.5 fold increase in in vivo target gene activation and a notable increase in the affinity for target DNA . The properties of the His50Cys mutant are discussed in terms of a variant structure of the DNA binding motif . During the course of this work, evidence was obtained suggesting that only one of the two LEU3 protein-DNA complexes routinely seen actually activates transcription . The other (which may contain an additional protein factor) does not.

Brain Res, 1991 Nov 8, 564(1), 79 - 85
Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells; Johnson CL et al.; This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UC11 . Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture . Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor . Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNF alpha) or interleukin-1 beta (IL1 beta), but not interleukin-2 or interleukin-6, decreased the density of SP receptors . This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation . The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines . The decrease in SP receptor density was readily reversible on washout of the cytokines . The EC50 for TNF alpha was approximately 0.5 ng/ml, the EC50 for IL1 beta was approximately 0.1 ng/ml . Radioligand binding studies with {125I}TNF alpha indicated the presence of a low density of high affinity binding sites for this ligand: Kd = 2.5 +/- 0.6 ng/ml, Bmax = 14.8 +/- 2.7 fmol bound/mg protein (assuming trimeric form of ligand bound) . The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.

Am J Physiol, 1991 Nov, 261(5 Pt 1), C822 - 7
Enhancement of kallikrein production and kinin sensitivity in T84 cells by growth in the nude mouse; Baird AW et al.; The production of tissue kallikrein and the short-circuit current (SCC) response in the human colonic epithelial cell line T84 to bradykinin have been studied . These cells produce true tissue kallikrein and secrete it into the growth medium, and addition of bradykinin results in a small increase in SCC . After growth of T84 cells as a xenograft in athymic (nude) mice (NuT84), however, the cellular concentration of the enzyme increases 38-fold, and the maximal change in SCC (delta SCC) induced by bradykinin increases 35-fold . The SCC response to prostaglandin E1 is not changed, and the response to forskolin increases eightfold . The bradykinin-induced delta SCC was inhibited by piretanide, suggesting that a secretory movement of chloride was responsible for the change, and the effect was attenuated by an antagonist to kinin receptors . We conclude that both the production of true tissue kallikrein and the chloride secretory response to bradykinin can be regulated in T84 cells by changes in the cell environment.

Infect Immun, 1991 Nov, 59(11), 3969 - 74
MDHM, a macrophage-stimulatory product of Mycoplasma fermentans, leads to in vitro interleukin-1 (IL-1), IL-6, tumor necrosis factor, and prostaglandin production and is pyrogenic in rabbits; Muhlradt PF et al.; Mycoplasma fermentans-derived high-molecular-weight material (MDHM) was originally discovered because of its capacity to generate, through the induction of monokine synthesis, cytolytic T lymphocytes in concanavalin A-stimulated thymocyte cultures . This study shows that MDHM-activated macrophages not only released interleukin-6 (IL-6) but also exhibited increased synthesis of cell-associated IL-1 as well as liberation of tumor necrosis factor and prostaglandin . We determined 6-keto prostaglandin F1 alpha since it is the stable metabolite of the bioactive prostacyclin . MDHM appeared to be as potent as lipopolysaccharide in inducing the synthesis of these mediators . Priming with gamma interferon further increased MDHM-mediated IL-6 release . Since monokines can be pyrogenic, we tested the effects of an intravenous injection of MDHM on rectal temperatures and leukocyte counts in rabbits . At 1 h after a bolus injection of MDHM, leukocyte counts dropped to about 35% of the initial values, reflecting a decrease in both lymphocytes and granulocytes . At 4 to 6 h after injection, granulocyte counts began to increase again, whereas lymphocyte counts remained low . No leukocytosis was noted during this time . The lack of leukocytosis can be explained by the failure of MDHM-stimulated macrophages to release IL-1 . The property of MDHM to cause IL-6 release from macrophages and the IL-6 growth dependency of the 7TD1 hybridoma cell line were made use of in a coculture assay system to quantitate the activity of MDHM . With this method and macrophages from C3H/HeJ lipopolysaccharide-nonresponder mice, MDHM activity was found to be equally distributed in the mycoplasma growth medium and the sedimented mycoplasmas after sonication.

Carcinogenesis, 1991 Nov, 12(11), 1993 - 9
Cell-to-cell communication: a differential response to TGF-beta in normal and transformed (BEAS-2B) human bronchial epithelial cells; Albright CD et al.; The effects of transforming growth factor beta (TGF-beta) on cell-to-cell communication were investigated in the log phase of growth in normal BE and in adenovirus-12 SV40 hybrid virus transformed BE cells (strain BEAS-2B) . Gap junctions in these cells were identified immunocytochemically . Exposure of BE cells to exogenous TGF-beta (0.04-4.0 pM) in serum-free keratinocyte growth medium (KGM) for 1 or 24 h reduced the rate of fluorescent dye transfer (i.e . cell-to-cell communication) by 30-50% in BE cells . Inversely, in BEAS-2B cells, TGF-beta after 1 h induced a 2- to 10-fold increase in the rate of dye transfer . After 24 h of TGF-beta, communication among BEAS-2B cells was not significantly different from controls (no exogenous TGF-beta) . The protein kinase C (PKC) inhibitor H-7 induced a dose-dependent enhancement in communication, which was even higher in the presence of TGF-beta (4 pM X 24 h) . The calmodulin antagonist W-7 enhanced communication in BEAS-2B cells independently of the presence of TGF-beta . In keratinocyte basal medium (KBM) supplemented with EGF (5 ng/ml) or with TGF-beta (4.0 pM) dye transfer was reduced or enhanced respectively . The combination of EGF and TGF-beta in KBM antagonized the stimulatory effect of the latter on communication in BEAS-2B cells . In BE cells, continuous exposure (4 days) to TGF-beta in KGM induced a dose-dependent inhibition of proliferation and an increased expression of a keratinized, epidermoid phenotype . This correlated with a reduction in the expression of a mucous secretory phenotype . Increased exposure to TGF-beta (0.04-4.0 pM) decreased the labeling index in BEAS-2B cells, but the cells retained a growth advantage over normal BE cells, and did not express a keratinized epidermoid morphology . With respect to dye transfer as an index of cell-to-cell communication, we conclude (i) that an inhibition or enhancement of communication is involved in the response of bronchial epithelial cells to mitogens (e.g . epidermal growth factor) or growth inhibitors (e.g . TGF-beta), (ii) that PKC and Ca(2+)-calmodulin-dependent processes regulate dye transfer, and (iii) the effects of TGF-beta are mediated by PKC.

Cancer Lett, 1991 Nov, 60(2), 109 - 12
Endogenous secretion of epidermal growth factor peptides stimulates growth of DU145 prostate cancer cells; Tillotson JK et al.; The growth rate of DU145 prostate cancer cells in vitro is slowed considerably by changing the growth medium every 24 h, suggesting dependence upon endogenously-secreted growth factors . Because previous studies have identified epidermal growth factor (EGF) in the conditioned medium from DU145 cells, {35S}labeled EGF was selectively immunoprecipitated from the culture medium at 24-h intervals for quantitation . Under the culture conditions used, there was an initial phase of slow growth, the EGF level secreted per cell was highest on day 3 after plating, and an increase in cell number was most evident between days 3 and 4 . Finally, growth was assayed under culture conditions where the medium was replaced every 24 h with fresh medium in the absence or presence of 10 ng/ml added EGF . The EGF was able to increase the cell growth up to the levels seen in cultures where the medium was unchanged during the entire period . We interpret these results as evidence that endogenously secreted EGF-like growth factors participate in an autocrine growth stimulation of DU145 cells.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 511 - 6
Regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae by inositol and choline: kinetics of repression and derepression; Overmeyer JH et al.; The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae (G . M . Carman and S . A . Henry, 1989, Annu . Rev . Biochem . 58, 635-669) . In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine . The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized . When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50-85% of control) within 60 min . After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15-40% of control values during a later stage of regulation (2-3 h) . In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2-3 h . The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol . In summary, the level of PS decarboxylase activity in S . cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline . The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).

J Protozool, 1991 Nov-Dec, 38(6), 613 - 23
A potential mucus precursor in Tetrahymena wild type and mutant cells; Ding Y et al.; By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide . We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved . This mutant contains no recognizable mature secretory granules (mucocysts) . By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products . Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter) . Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa . Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell . Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells . Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively . These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.

Immunol Lett, 1991 Nov, 30(3), 325 - 32
Properties of human pre- and post-implantation embryo-associated immunosuppressor factor(s); Bose R; Embryo-associated immunosuppressor factor (EASF), a factor detected by its suppressive property on the concanavalin A (ConA) stimulated lymphocyte proliferation assay, was purified from human embryo growth media of in vitro fertilized ova (pre-implantation source) and from pregnancy sera (post-implantation source) as 3 fractions, CM-1, CM-3 and CM-6, the immunosuppressive properties of which were studied . The results show that, (i) the 3 fractions of EASF from both sources suppressed ConA- and pokeweed mitogen (PWM)-induced lymphocyte proliferation, suggesting that they have suppressive effects on both T and B cells; (ii) all 3 EASFs were suppressive when added at the early phase of ConA-supplemented cultures; (iii) CM-1 of both sources were suppressive when added to PWM-supplemented cultures between 24 and 48 h; and (iv) CM-6 of both sources showed an irreversible immunosuppressive effect on PWM-induced lymphocyte proliferation, demonstrating that some similarities exist in the immunosuppressive property of EASF from the 2 sources . On the other hand, (i) CM-6 of pre- and post-implantation EASF were immunosuppressive when added to the PWM-supplemented cultures at 24-48 h and 0-16 h, respectively; and (ii) the CM-6 fraction of pregnancy sera, but not the CM-6 fraction of embryo growth media, possessed an irreversible immunosuppressive effect on ConA-supplemented cultures . This active process by which EASF affects T cell and B cell functions directly may be one of several responses by which the maternal immune response against the fetus is prevented.

Yeast, 1991 Nov, 7(8), 849 - 55
The cysteine transport system of Saccharomyces cerevisiae; Ono B et al.; Although Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT (83.3 microM) . The optimal pH of cysteine uptake was between 4.5 and 5.0, but the activity was quickly lost if cells were kept in buffer . When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride . Thioglycol as well as metabolic inhibitors such as dinitrophenol and azide were inhibitory . Homocysteine and methionine were competitive and non-competitive inhibitors, respectively . Cysteamine and cysteic acid were not inhibitory . From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH-group but also amino- and carboxyl-groups . In wild-type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source . On the other hand, in cysteine-dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine . From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains.

Methods Find Exp Clin Pharmacol, 1991 Nov, 13(9), 595 - 8
Growth medium for the evaluation of antiestrogenic compounds in MCF-7 cell culture; Jain PT et al.; The primary aim of the present study was to define a set of cell culture conditions that would be optimal for the evaluation of antiestrogens using estrogen dependent MCF-7 human breast cancer cells in culture . Therefore, the present study examined for the influence of media supplements which are known to alter the growth rate and estrogen receptor content of MCF-7 cells in culture . In this study, MCF-7 cell proliferation was evaluated using the hemocytometric trypan blue exclusion method . The results of this study clearly indicate that 1) charcoal-dextran stripped serum offers no advantage over low estradiol (less than 100 fM) calf serum which is available commercially, and 2) calf serum is as effective as fetal calf serum, in the presence or absence of insulin, in time-dependent growth of MCF-7 cells . Thus, growth media containing 5% non-stripped low estradiol (less than 100 fM) calf serum without insulin supplementation was found to be optimal for the evaluation of the antitumor activity of antiestrogenic compounds using MCF-7 cells in culture.

Anticancer Res, 1991 Nov-Dec, 11(6), 1995 - 8
The role of ethanol in oncogenesis of the upper aerodigestive tract; inhibition of DNA repair; Hsu TC et al.; When cells of four lymphoblastoid lines were pulse treated with bleomycin for 10 min and reincubated in control medium or medium containing 0.5% ethanol, the frequencies of chromatid breaks steadily dropped as incubation time increased . At 5 hr after the pulse, the break/cell rate was reduced to a level slightly higher than that of the untreated cell populations . When the cells were reincubated in medium containing 2% ethanol, the chromatid breakage rates remained high throughout the 5-hr period . Apparently DNA repair processes were inhibited by 2% ethanol . However, this inhibition phenomenon could be reversed when ethanol was removed from the growth medium . The finding that ethanol inhibits DNA repair helps explain why ethyl alcohol has been regarded as a cocarcinogen in head and neck cancers.

Mol Biochem Parasitol, 1991 Nov, 49(1), 21 - 8
Biopterin conversion to reduced folates by Leishmania donovani promastigotes; Beck JT et al.; The ability of Leishmania donovani promastigotes to proliferate in folate-deficient medium supplemented with pterins suggests that pterins can serve as a source of folate in these parasites {16} . Using reversed-phase high-performance liquid chromatography, the ability of intact L . donovani to transform {3H}biopterin into tetrahydrofolates was demonstrated . Radioactivity was primarily associated with 5-methyltetrahydrofolate and 10-formyltetrahydrofolate . A mutant strain of L . donovani, MTXA5, that was genetically deficient in folate transport capacity and incapable of growing in pterin-supplemented folate-deficient growth medium, exhibited a greatly reduced capacity to metabolize {3H}biopterin to reduced folates . These data indicated that wild-type L . donovani promastigotes, unlike mammalian cells, were able to convert biopterin to tetrahydrofolates and supported the hypothesis that folate transport deficiency in mutant organisms is associated with an inability to transform pterins to reduced folates.

Plasmid, 1991 Nov, 26(3), 186 - 200
Maintenance of pBR322-derived plasmids without functional RNAI; Chiang CS et al.; pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed . Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter . The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65) . Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication . The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication . In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations . During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG . As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases . This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it . It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9680 - 4
Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts; Goldstein S et al.; Insulin-like growth factor binding protein 3 (IGFBP-3) mRNA levels were consistently higher in both senescent normal human diploid fibroblasts (HDFs) at late passage (old cells) and prematurely senescent HDFs from a subject with Werner syndrome (WS) during serum depletion and repletion of growth medium and during proliferation from sparse to high-density inhibited cultures, compared to normal early-passage (young) HDFs . However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions . Insulin-like growth factor I (IGF-I) was not detected in naive medium or in any of the media conditioned by these three cell types, whereas IGF-II was detectable in serum-repleted medium and remained relatively constant . Thus, molar ratios of IGFBP-3/IGF-II were consistently higher in old and WS cells and increased substantially as all three cell types became quiescent, due to either serum depletion or high cell density . These data are consistent with either an adaptive or a causal role for IGFBP-3 protein in the senescent and quiescent growth arrest of HDFs.

J Invertebr Pathol, 1991 Nov, 58(3), 408 - 14
Effects of exogenous phospholipids on lipid composition and sporulation by three strains of Lagenidium giganteum; Kerwin JL et al.; The California (LGCA) and Butte Sink (LGBS) strains of the sterol auxotrophic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) enter the sexual cycle on media supplemented with sterols . A third isolate of this mosquito pathogen, the North Carolina strain (LGNC), requires sterols plus phospholipids to produce oospores in vitro . Enrichment of the polar and neutral lipid fractions of the LGCA and LGBS strains with unsaturated fatty acids promoted oospore induction, and increased oospore viability . With the exception of the LGCA strain, there was no consistent relationship between phospholipid supplementation in growth media and mycelial phospholipid content.

J Biotechnol, 1991 Nov, 21(1-2), 21 - 42
Effect of culture process on alkaloid production by Catharanthus roseus cells . II . Immobilized cultures; Tom R et al.; Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated . The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d . The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM . The production stage which lasted for 15 d . High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation . Growth, main nutrient consumption and alkaloid production were monitored . Both culture regimes resulted in similar biomass production, dw (10-13 g l-1) . The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%) . Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures . More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1) . These results were compared to those obtained from shake flask cultures performed at the same time, with the same C . roseus cell line and under similar regimes and reported previously . Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production . However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures . Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures . Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions . Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.

J Biotechnol, 1991 Nov, 21(1-2), 109 - 25
Production of hepatitis B surface antigen (preS2 + S) by high-cell density cultivations of a recombinant yeast; Schulman CA et al.; A high-cell density bioprocess has been developed for the production of hepatitis B surface protein (preS2 + S) by recombinant yeast . This fed-batch process utilizes a growth medium containing yeast extract, soy peptone and glucose which was fed at a constant rate to maintain cells in a respiratory state . Cell densities of up to 60 g l-1 dry weight were achieved, which represented a 6-fold increase over those from batch bioprocesses . This increase in cell mass was attained without compromising specific activity; therefore, volumetric productivities of six times those of batch bioprocesses were achieved.

J Biotechnol, 1991 Nov, 21(1-2), 1 - 19
Effect of culture process on alkaloid production by Catharanthus roseus cells . I . Suspension cultures; Tom R et al.; The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated . The 1-stage process consisted of inoculating APM and incubating for 15 days . The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM . Growth, main nutrient consumption and alkaloid production were monitored . Both culture processes produced approximately 20 g dw per 1 biomass . However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (greater than 70%) nutrient availability and consumption . Total and individual indole alkaloid production were 10 times higher (740 mg l-1 and 25 to 4000 micrograms per g dw, respectively) for 2-stage than for 1-stage cultures . For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells . Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in production . Small but significant (approximately 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.

Gene, 1991 Oct 30, 107(1), 149 - 54
Cloning and transcriptional analysis of the ADE6 gene of Saccharomyces cerevisiae; Giani S et al.; The Saccharomyces cerevisiae gene, ADE6, encoding 5'-phosphoribosylformyl glycinamidine synthetase (EC 6.3.5.3) has been cloned by complementation of an ade6 auxotroph . Transformation of ade6 mutants with ADE6-carrying centromeric plasmids restored normal, adenine-independent growth behavior in the recipients . Strains containing a disrupted ade6 allele were constructed and behaved as stable adenine auxotrophs . Southern transfer and genetic analyses of strains carrying a disrupted ade6 allele demonstrated that the cloned gene was ADE6 and not a suppressor . The cloned ADE6 DNA was mapped on the RAD2-proximal fragment of chromosome VII by hybridization on yeast chromosomes separated by pulsed-field gel electrophoresis . Northern-blot hybridization experiments show that the ADE6 region produces two different mRNA species of approx . 5 and 2 kb . Disappearance of the larger, but not the smaller, transcript is associated with ade6 mutations . A threefold repression in the amount of the 5-kb ADE6 mRNA is observed when growth medium is supplemented with exogenous adenine.

J Biol Chem, 1991 Oct 15, 266(29), 19758 - 67
Chitinase is required for cell separation during growth of Saccharomyces cerevisiae; Kuranda MJ et al.; The Saccharomyces cerevisiae chitinase described by Correa et al . (Correa, J . U., Elango, N., Polacheck, I., and Cabib, E . (1982) J . Biol . Chem . 257, 1392-1397) has been cloned and sequenced . Analysis of the derived amino acid sequence suggests that the protein contains four domains: a signal sequence, a catalytic domain, a serine/threonine-rich region, and a carboxyl-terminal domain with high binding affinity for chitin . Most of the enzyme produced by cells is secreted into the growth medium and is extensively glycosylated with a series of short O-linked mannose oligosaccharides ranging in size from Man2 to Man5 . Chitinase O-mannosylation was further examined in the temperature-sensitive secretion mutants sec18, sec7, and sec6 . Oligosaccharides isolated from chitinase accumulating in cells at the nonpermissive temperature revealed Man1 and Man2 associated with the sec18 mutant . sec6 and sec7 accumulated Man2-Man5 with a higher proportion of Man5 relative to the secreted protein . A significant amount of chitinase is also found associated with the cell wall through binding of COOH-terminal domain to chitin . Disruption of the gene for the enzyme leads to a defect in cell separation but does not substantially alter the level of cellular chitin.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 311 - 6
pSAR1, a natural plasmid from Streptomyces arenae, shows rapid increase and decrease of copy numbers on changes of growth media; Braxenthaler M et al.; A natural plasmid, pSAR1, was isolated from the antibiotic producer Streptomyces arenae TU469 . Its size is estimated to approx . 80 kbp by restriction analysis . pSAR1 occurs in two copy-number states differing by a factor of at least 10, depending on culture conditions . The high copy-number state is strongly correlated with the production of the antibiotic pentalenolactone . The decrease of copy numbers after change of culture conditions is completed within 1 h . These unusually rapid kinetics and the occurrence of degradational intermediates suggest the participation of specific catalytic mechanisms in copy number regulation.

J Biol Chem, 1991 Sep 25, 266(27), 17863 - 71
Insertion of the mannitol permease into the membrane of Escherichia coli . Possible involvement of an N-terminal amphiphilic sequence; Yamada Y et al.; The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches . Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro . The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses . Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage . Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed . In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase . The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence . The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm . This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment . Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation . The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis . The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly... . The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1991 Oct, 173(20), 6515 - 27
Components of ice nucleation structures of bacteria; Turner MA et al.; Nonprotein components attached to the known protein product of the inaZ gene of Pseudomonas syringae have been identified and shown to be necessary for the most efficient ice nucleation of supercooled H2O . Previous studies have shown that cultures of Ina+ bacteria have cells with three major classes of ice-nucleating structures with readily differentiated activities . Further, some cells in the culture have nucleating activities intermediate between those of the different classes and presumably have structures that are biosynthetic intermediates between those of the different classes . Since these structures cannot be readily isolated and analyzed, their components have been identified by the use of specific enzymes or chemical probes, by direct incorporation of labeled precursors, and by stimulation of the formation of specific classes of freezing structures by selective additions to the growth medium . From these preliminary studies it appears that the most active ice nucleation structure (class A) contains the ice nucleation protein linked to phosphatidylinositol and mannose, probably as a complex mannan, and possibly glucosamine . These nonprotein components are characteristic of those used to anchor external proteins to cell membranes of eucaryotic cells and suggest that a similar but not identical anchoring mechanism is required for efficient ice nucleation structure . The class B structure has been found to contain protein presumably linked to the mannan and glucosamine moieties but definitely not to the phosphatidylinositol . The class C structure, which has the poorest ice nucleation activity, appears to be the ice nucleation protein linked to a few mannose residues and to be partially imbedded in the outer cell membrane.

J Bacteriol, 1991 Oct, 173(19), 6009 - 17
Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli; Yanofsky C et al.; Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB . The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation . We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins . In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction . High levels are necessary because much of the added tryptophan is degraded by tryptophanase . An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants . In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction . However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction . In this medium, AroP does not contribute to tryptophan uptake . However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction . The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action . The TnaB permease is essential for growth on tryptophan as the sole carbon source . When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated . This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency . Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions.

Br J Cancer, 1991 Oct, 64(4), 671 - 6
Phorbol ester and bryostatin effects on growth and the expression of oestrogen responsive and TGF-beta 1 genes in breast tumour cells; Nutt JE et al.; The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) (10 nM) produce a marked reduction in the growth, measured by thymidine uptake, of MCF-7 cells in full growth medium, but had only a small effect on MDA-MB-231 and T47D cells . Bryostatin alone also inhibited growth but to a lesser extent than seen with TPA . The effect of TPA on MCF-7 cells was partially reversed by bryostatin, added simultaneously or after TPA, suggesting bryostatin does not simply mimic TPA in this system . Even though both are believed to act via effects on protein kinase C, bryostatin appears to act as antagonist to the effect of TPA as well as a partial agonist on its own . When the oestrogen receptor positive MCF-7 and T47D cells were maintained in charcoal stripped serum, the increase in DNA synthesis on stimulation with oestradiol was inhibited with 50 nM TPA in MCF-7 cells but not in T47D cells . The effects of these treatments on the expression of two well characterised oestrogen responsive genes pNR2(pS2) and pNR100 (Cathepsin-D) were examined . Rather than preventing transcription of these oestrogen responsive genes, TPA alone increased pNR2 and pNR100 levels in MCF-7 cells and the combined effect of oestradiol and TPA had a marked synergistic effect in increasing the transcript levels of these genes . In T47D cells pNR2 transcripts were not detected and the increase in pNR100 mRNA levels were not affected by TPA . We conclude that the inhibitory effects of TPA on the growth stimulation of MCF-7 cells by oestradiol was not due to a general inhibition of the expression of oestrogen responsive genes . An alternative possibility examined was that the growth inhibitory effect of TPA on MCF-7 cells might be due to stimulation of TGF-beta 1, acting as an autocrine inhibitory growth factor . Oestradiol treatment of MCF-7 cells reduced the levels of TGF-beta 1 mRNA whereas TPA produced a marked increase . The combined effect of TPA and oestradiol further increased TGF-beta 1 mRNA above the levels seen with TPA alone . Bryostatin had little effect on TGF-beta 1 expression either alone or in combination with oestradiol . These observations are consistent with the hypothesis that the inhibitory effect of TPA on MCF-7 cells may be partly due to autocrine inhibition by TGF-beta 1.

Br J Cancer, 1991 Oct, 64(4), 663 - 70
Glucose starvation and acidosis: effect on experimental metastatic potential, DNA content and MTX resistance of murine tumour cells; Schlappack OK et al.; Exposure to oxygen deprivation in vitro has been reported to cause drug resistance in CHO cells (Rice et al., 1986; PNAS 83, 5978) and enhancement of experimental metastatic (colonisation) ability of murine tumour cells (Young et al., 1988; PNAS 85, 9533) . Both these studies also demonstrated the induction of a subpopulation of cells with excess DNA content . Since the micromilieu in tumours results in exposure of the tumour cells to conditions of acid pH and nutrient deprivation, as well as hypoxia, we have examined the effect of exposure to acidosis (pH 6.5) and glucose starvation on drug resistance, cellular DNA content and the experimental metastatic ability of KHT sarcoma and B16F1 melanoma cells . Cells were exposed to these conditions for 24 and 48 h and tested for resistance to methotrexate (MTX) or experimental metastatic ability either immediately following these exposures or after 24 or 48 h of recovery in normal growth medium . Both cell lines demonstrated an enhancement of colonisation potential, which was most marked when cells were injected after 48 h of exposure followed by a 24 or 48 h recovery period . Flow cytometric analysis demonstrated an increase in the fraction of KHT cells with excess DNA following both glucose starvation and acidosis we observed only a small increase in MTX resistance following acidic exposure of cells and no change following glucose starvation . Since both acidosis and glucose starvation are known to induce glucose regulated proteins (grp), a subset of the stress protein family, we studied the effect of treatment with another known inducer, 2-deoxyglucose . We found that this agent affected the metastatic efficiency of KHT cells in a manner similar to that observed following exposure to glucose starvation and acidosis . However, further studies are required to establish what role, if any, grp play in this effect . In conclusion this study shows that transient exposure of murine tumour cells to an acidic or glucose deprived environment can cause progression in terms of metastatic potential.

Res Commun Chem Pathol Pharmacol, 1991 Oct, 74(1), 105 - 16
A vehicle for the evaluation of hydrophobic compounds in cell culture; Jain PT et al.; A vehicle for testing hydrophobic compounds in cell culture has been evaluated on human breast (MCF-7 and MDA-MB-231) and human lung (A 549) cancer cells . The cytotoxicity of the vehicles was evaluated on the basis of cell viability using the hemocytometric trypan blue exclusion method and cell surface morphology using scanning electron microscopy . Dimethylacetamide, absolute ethanol and polyethylene glycol 400 were evaluated as vehicles for solubilizing various hydrophobic compounds . The compounds were found to be most soluble in dimethylacetamide but this solvent was extremely cytotoxic to MCF-7 cells . Similarly, 1% absolute ethanol solubilized the compounds in culture media at a concentration of 10(-5)M, but was also cytotoxic . 0.1% absolute ethanol in the culture media was non-cytotoxic but was unable to solubilize these hydrophobic compounds . Polyethylene glycol 400 was found to be too viscous to use alone . However, upon optimizing the ratio of absolute ethanol and polyethylene glycol 400, a mixture of 45% absolute ethanol plus 55% polyethylene glycol 400 at a final concentration of 0.1% was used as vehicle solubilized the hydrophobic compounds (10(-5)M) in growth media and non-cytotoxic on all the cell lines tested . In conclusion, a vehicle containing a mixture of 45% absolute ethanol plus 55% polyethylene glycol 400 at a final concentration of 0.1% of the growth medium was found to be ideal for testing various hydrophobic compounds in cell culture.

J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2331 - 7
Plasmid effects on secondary metabolite production by a streptomycete synthesizing an anthelmintic macrolide; Thomas DI et al.; Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity) . However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains . A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before . All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium . Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used . In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton . The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production . No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

J Neurosci Res, 1991 Oct, 30(2), 308 - 15
Myelin basic protein specific T cell lines and clones derived from the CNS of rats with EAE only recognize encephalitogenic epitopes; Bourdette DN et al.; The epitope specificities of myelin basic protein (BP) specific T cell lines derived from the spinal cords (SC) and lymph nodes (LN) of rats with experimental autoimmune encephalomyelitis (EAE) were compared . To induce EAE, Lewis rats were immunized with guinea pig (GP)-BP and complete Freund's adjuvant . Mononuclear cells from the SC and LN of these animals proliferated in response to BP and the purified protein derivative (PPD) of mycobacterium . After initially being cultured in growth medium, SC mononuclear cells had an enhanced response to BP and lost their response to PPD . LN cells cultured in identical conditions lost their response to both BP and PPD . LN-derived BP specific cell lines recognized only two epitopes of GP-BP: an encephalitogenic epitope in residues 72-89 and a non-encephalitogenic epitope in residues 43-68 . SC-derived BP specific cell lines and clones recognized the 72-89 epitope and a second encephalitogenic epitope contained in residues 87-99 but not the non-encephalitogenic 43-68 epitope . Unlike those from LN, BP-specific T cell lines and clones derived from the CNS appear to recognize only encephalitogenic epitopes, including epitopes not recognized by LN lines.

Antonie Van Leeuwenhoek, 1991 Oct-Nov, 60(3-4), 225 - 34
On multiple-nutrient-limited growth of microorganisms, with special reference to dual limitation by carbon and nitrogen substrates; Egli T; Simultaneous limitation of microbial growth by two or more nutrients is discussed for dual carbon/nitrogen-limited growth in continuous culture . The boundaries of the zone where double-limited growth occurs can be clearly defined from both cultivation data and cellular composition and they can be also predicted from growth yield data measured under single-substrate-limited conditions . It is demonstrated that for the two nutrients carbon and nitrogen the zone of double nutrient limitation is dependent on both the C:N ratio of the growth medium and the growth (dilution) rate . The concept on double-(carbon/nitrogen)-limited growth presented here can be extended to other binary and multiple combinations of nutrients.

Br J Pharmacol, 1991 Oct, 104(2), 419 - 27
Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture; Griffiths NM et al.; 1 . Human cells (HeLa) were cultured for periods up to 48 h in growth medium in the absence or presence of a range of concentrations of cardiac glycosides . In some experiments the potassium concentration of the medium was varied between 0.3 mM and the usual 5 mM . 2 . For periods up to 2 h in ouabain the association and dissociation rate constants were measured and the equilibrium binding constant (KD) calculated; the apparent equilibrium binding constant (K'D) was measured after 1-2 days growth in ouabain . 3 . Ouabain had a K'D after 2 days of 2-6 nM in 5 mM K+ growth medium, a 4 fold greater blocking effect on sodium pumps after 2 days than expected from the association and dissociation rate constants measured in untreated or previously ouabain-treated cells . 4 . This effect was: (a) approximately the same over a range of external potassium concentrations from 0.3 to 5 mM, although the absolute effect of ouabain over this range of potassium was much different; (b) probably not due to different isoforms of pumps in cells grown in ouabain compared to untreated cells; (c) apparently not a consequence of internalisation of pump-glycoside complexes . 5 . We conclude that ouabain has only a limited access to sodium pumps in whole cells; this could be because sodium pumps cycle continuously through an inaccessible region of the plasma membrane . This effect needs to be considered both in the assessment of the magnitude of the long term effects of cardiac glycosides on cells, and in the measurement of the glycoside affinities of various isoforms of the pump.

FEMS Microbiol Lett, 1991 Sep 15, 67(1), 23 - 8
Secretion of oligomeric Val8-human calcitonin by Saccharomyces cerevisiae; Mironova R et al.; Monomeric human calcitonin (hCT) gene and oligomeric hCT genes composed of two, three or four head-to-tail linked monomers were fused in-frame to the yeast alpha-factor leader coding sequence wild-type and fragile mutant Saccharomyces cerevisiae strains were transformed with the constructed plasmids and the yield of recombinant protein secreted into the culture medium was measured . The yeast cells secreted equal (molar) amounts of all of the hCT variants . The recombinant proteins remained stable in the growth medium for at least 3 days . The fragile cells secreted about 30% more hCT as compared to the wild-type yeast cells.

J Biol Chem, 1991 Sep 5, 266(25), 16273 - 6
Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake; Sturley SL et al.; We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae . Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol . Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol . ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media . This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions . Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction . Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2) . The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells . One such factor could be the mammalian equivalent of the gene product of UPC2.

Can J Microbiol, 1991 Sep, 37(9), 718 - 21
Extracellular release of protease III (ptr) by Escherichia coli K12; Diaz-Torres MR et al.; Escherichia coli strains carrying the protease III structural gene (ptr) on a plasmid secreted the protein into the growth medium . Plasmid-encoded beta-lactamase and chloramphenicol acetyl transferase, which served as periplasmic and cytoplasmic markers during cell fractionation, were not released into the growth medium . There appeared to be some strain dependence on the proficiency of the secretion system . Protease III was not detectably processed upon export through the outer cell membrane.

Arch Environ Contam Toxicol, 1991 Sep, 21(3), 425 - 31
Binding of cadmium by cyanobacterial growth media: free ion concentration as a toxicity index to the cyanobacterium Nostoc UAM 208; Fernandez-Pinas F et al.; A quantitative study of cadmium binding to three different growth media for nitrogen-fixing cyanobacteria was done with the aid of a solid state ion-specific electrode . Kratz and Myers modified medium and Arnon medium bound large amounts of Cd2+, BG11o medium had less binding capacity . Of the media components, phosphate ion showed the greatest ability to bind Cd2+ . Different pHs, the size of cell inoculum and two buffers (Tricine and HEPES, 25 mM) also changed the availability of free cadmium ion in solution . The effect of free Cd2+ ion towards the cyanobacterium Nostoc UAM 208, isolated from a heavy metal polluted environment, also was tested . The effective concentration affecting 50% of population (EC50), at 120 h of exposure, was less for nitrogenase activity (0.26 microgram/mL) than for growth (0.55 microgram/mL), suggesting that this enzyme activity is more sensitive to cadmium than growth . Furthermore, cadmium toxicity was influenced by the addition of buffers to the growth medium . In the presence of buffer, Tricine (25 mM), growth and nitrogenase activity was reduced by 50% at a total cadmium concentration of about 115 micrograms/mL, although no free ion was detected in this case . These results suggest that although generally cadmium toxicity is a function of free metal ion concentration, this can also vary in the presence of complexing agents.

Mol Gen Genet, 1991 Sep, 228(3), 410 - 6
Isolation and genetic analysis of a mutation that suppresses the auxotrophies of superoxide dismutase-deficient Escherichia coli K12; Imlay JA et al.; The most striking phenotype associated with superoxide dismutase (SOD) deficiency in Escherichia coli is the inability to grow in aerobic minimal medium, which is due to the sensitivity of several amino acid biosynthetic pathways to superoxide . We have isolated two classes of pseudorevertants that grow on minimal medium at modest rates . Of these, the class that exhibited the faster growth carries mutations at a single locus, denoted ssa, which was mapped to 4 min on the E . coli chromosome . This class constituted the majority of the spontaneous pseudorevertants that were selected by the transfer of independent SOD-deficient cultures in minimal medium from anaerobic to aerobic growth conditions . Pseudoreversion at ssa suppressed requirements for a variety of unrelated amino acid supplements . Further, the SOD-deficient strains were unable to assimilate diaminopimelic acid from the growth medium, whereas the ssa pseudorevertants did so . The viability of these pseudorevertants indicates that superoxide-sensitive biosynthetic enzymes do retain some function in SOD-deficient cells during aerobic growth.

Curr Eye Res, 1991 Sep, 10(9), 851 - 63
Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro; He YG et al.; Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro . Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM) . We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages . With this combined medium, a suspension of cultured corneal epithelial cells (5 x 10(5)/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel or type IV collagen) . The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached . However, the cells on the shields coated with Matrigel failed to become confluent under these conditions . The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days . After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.

Nihon Kyobu Shikkan Gakkai Zasshi, 1991 Sep, 29(9), 1143 - 9
{Proliferative characteristics of fibroblast lines derived from open lung biopsy specimens of patients with the usual interstitial pneumonia form of idiopathic pulmonary fibrosis}; Mio T; One form of idiopathic pulmonary fibrosis (IPF), usual intersitial pneumonia (UIP) is characterized pathologically by patchily distributed fibrotic areas in apparently normal parenchyma . Excessive accumulation of collagen and fibroblasts in fibrotic areas are shown histologically . Fibroblast proliferation is generally evaluated as a process following alveolitis . However, substantial alveolitis with increased inflammatory and immune cells were not observed in our UIP cases . To evaluate the possibility that fibroblasts in UIP are controlled by mechanisms other than normal paracrine regulation, proliferative features of lung fibroblast lines from UIP lung with regular growth medium, platelet derived growth factor (PDGF) and prostaglandin E2 (PGE2) were investigated . Ten fibroblast lines from open lung biopsy specimens of patients with IPF (UIP) and 10 control fibroblast lines from surgically resected lung tissues of patients with limited lung disease were established . The doubling time of fibroblast lines with regular growth medium was UIP:32.0 +/- 6.0 hrs . (mean +/- S.D.), normal control: 33.2 +/- 10.4 hrs . There was no difference between the groups . To examine growth promotion activity by PDGF and growth inhibition by PGE2, lung specimens from 4 patients with IPF were subdivided into tissue with high intensity fibrotic lesion (H) and low intensity fibrotic lesion (L), and the fibroblast lines were established separately . 3H-thymidine uptake with or without PDGF and PGE2 was examined, and results were expressed as the stimulation index . Growth promotion by PDGF was H: 1.97 +/- 1.19, L: 1.89 +/- 0.78, normal control: 2.29 +/- 0.55 . There were no differences between groups . Growth inhibition by PGE2 was H: 0.88 +/- 0.24, L: 0.69 +/- 0.49, normal control: 0.44 +/- 0.33 . Growth inhibition for H was significantly lower than control (p less than 0.05) . Growth inhibition for L was lower than controls, but the difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vitro Cell Dev Biol, 1991 Sep, 27A(9), 749 - 54
Cultured proliferating rat mammary epithelial cells; Ehmann UK et al.; Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line . Proliferation of the normal rat cells occurred as the LA7 cells slowly died from the radiation . By labeling the cultures with 3H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells . The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells . If the cells were plated at a ratio of approximately 1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk . The cells have so far been carried up through Passage 7, which amounted to approximately 19 doublings in cell number, and still proliferate vigorously . The growth medium for this culture system was Dulbecco's modified Eagle's medium:Ham's F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics . The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin . The cultures were composed of cells with diploid or near diploid chromosome numbers . Samples of the cultured cells were implanted into the cleared fat pads of nude mice . Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no outgrowths resulted from the implantation of cells from Passage 5 . The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads.

Mol Gen Genet, 1991 Sep, 229(1), 96 - 108
Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae; Korch C et al.; The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP:adenylylsulfate-3'-phosphotransferase, EC 2.7.1.25), has been cloned . The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23,060 dalton . Translational fusions of MET14 with the beta-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca . 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium . By primer extension the MET14 transcripts were found to start between positions -25 and -45 upstream of the initiator codon . Located upstream of the MET14 gene is a perfect match (positions -222 to -229) with the previously proposed methionine-specific upstream activating sequence (UASMet) . This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PHO5 gene to which the yeast protein PHO4 binds . The human oncogenic protein c-Myc also has the same recognition sequence . Furthermore, in the 270 bp upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element . The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E . coli and chromosomally integrated . We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively . We propose that both types of control are exerted on MET14.

Eur J Biochem, 1991 Aug 15, 200(1), 113 - 22
Regulation of transcription of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase of Saccharomyces cerevisiae; Einerhand AW et al.; Transferring Saccharomyces cerevisiae cells from glucose- to oleate-containing growth media results in a significant increase in the number and volume of peroxisomes . To investigate this proliferation process we studied the transcriptional regulation of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase (EC 2.3.1.16) in response to the switch in carbon source . Expression was proved to be repressed during growth on glucose, derepressed during growth on glycerol, and induced during growth on oleate as the sole carbon source . By deletion and mutational analysis of sequences upstream of this gene, we have identified a region which is involved in the regulation of transcription . It is contained within a 52-base-pair sequence, UAST52 (upstream activation sequence thiolase 52), located between 203 and 151 nucleotides upstream of the translational initiation codon . This sequence proved to be required for repression, derepression and induction of transcription, and was able to activate transcription from the truncated version of the heterologous iso-1-cytochrome-c (CYC1) promoter in a similar way as in the wild-type promoter context . Sequence comparison revealed that the UAST52 contained a sequence motif ('beta-oxidation box') that is very similar to sequences located in the 5'-upstream regions of the genes coding for two other beta-oxidation enzymes of S . cerevisiae: the peroxisomal acyl-CoA oxidase and the peroxisomal trifunctional beta-oxidation enzyme of S . cerevisiae . Mutational analysis of the 'beta-oxidation box' indicates that this sequence motif acts as a UAS in vivo . Sequence comparison also revealed that just upstream of the 'beta-oxidation box', between positions -213 and -201, a potential binding site occurred for the yeast multifunctional autonomously replicating sequence binding factor ABF1 . Gel-retardation-competition experiments indicate that ABF1 binds specifically to this sequence.

J Biol Chem, 1991 Aug 5, 266(22), 14198 - 201
Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast; Reppe S et al.; Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Hogseth, A., Blingsmo, O . R., Saether, O., Gautvik, V . T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O . S., Gordeladze, J . O., Alestrom, P., and Gautvik, K . M . (1990) J . Biol . Chem . 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O . S., Reppe, S., Saether, O., Blingsmo, O . R., Sletten, K., Gordeladze, J . O., Hogset, A., Gautvik, V . T., Alestrom, P., Oyen, T . B., and Gautvik, K . M . (1990) Gene (Amst.) 90, 255-262) . In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27) . To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S . cerevisiae as a fusion protein with the alpha-factor leader sequence . The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing . Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide . Using improved purification procedures, a significantly higher purity was also obtained . The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry . Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.

Antonie Van Leeuwenhoek, 1991 Aug, 60(2), 67 - 72
Cultural conditions for Aeromonas hydrophila affect the production of haemolysins with differing host specificities; Nzeako BC et al.; Five strains of Aeromonas hydrophila were studied for production of haemolysin specific for erythrocytes of various animal species using three cultural methods . All the strains produced haemolysin for all the erythrocyte species when the organisms were cultured on blood agar . Using cellophane overlay method, all the strains produced haemolysin for fish erythrocytes and variable activity to mammalian erythrocytes . Only one strain produced haemolytic activity for various though not all of the erythrocyte species when grown in brain heart infusion broth . Data suggest that A . hydrophila produces multiple haemolysins with specificities for erythrocytes of different animals . This was confirmed for trout and horse erythrocyte targeted haemolysins, by using iso-electric focussing separation and by measuring the effect of addition of ammonium sulphate to the growth medium.

Lipids, 1991 Aug, 26(8), 598 - 603
Involvement of heme components in sterol metabolism of Saccharomyces cerevisiae; Lorenz RT et al.; There is an intimate association between sterol biosynthesis in yeast and aerobicity . Besides the requirement for molecular oxygen for the epoxidation of squalene, cytochrome hemoproteins are involved in demethylation and desaturation steps . Regulatory effects of hemes on sterol formation have been demonstrated using specifically defective mutants of yeast . Heme competency participates in a mechanism whereby wild-type cells are prevented from taking exogenous sterols from the growth media . The multiple interactions of hemes and sterols appear to be associated with the variously defined functions for sterols in the yeast cells.

Rinsho Shinkeigaku, 1991 Aug, 31(8), 809 - 14
{Effects of A23187 and cytochalasin B on proliferation and differentiation of the cultured myoblasts}; Sato K et al.; The effects of A23187 (a substance acting on cell membrane) and cytochalasin B (a substance acting on cell structure) on the growth and differentiation of myoblasts (L6 cell) were examined using a cultured system . The L6 cells (1 x 10(5)) were incubated for 10 days in DMEM medium containing 10% fetal calf serum (a growth medium) by exchanging the medium every 3rd day . They showed no morphological differentiation and no increase in creatine kinase activity, myoglobin content and Ca2+ concentration . However, when L6 cells were incubated in DMEM medium containing 2% horse serum and 6 micrograms/ml insulin (an incubation medium for differentiation), they were morphologically differentiated into the myotube after 6-day culture with increase in creatine kinase activity, myoglobin content and Ca2+ concentration . In the presence of 40 nM A23187, no marked morphological change was observed in the L6 cells as compared with the control, but the myoglobin level rapidly increased to 18.2 x 10(-2) ng/micrograms (4.2-fold value of the control) at 10-day culture . In the presence of 200 nM cytochalasin B, there was no morphological change in the L6 cells, and no increase in the levels of creatine kinase and myoglobin . These data suggest that the function of the cell membrane and intracellular Ca2+ concentration play important roles in differentiating the muscle cells, because A23187 promotes biochemical differentiation of L6 cells as shown by increased myoglobin content.

Biol Trace Elem Res, 1991 Aug, 30(2), 145 - 62
Effect of selenium compounds and thiols on human mammary tumor cells; Yan L et al.; The effect on cell viability and growth rate of sodium selenite, selenocystine, sodium selenate, and selenomethionine at selenium concentrations of 6.25 and 12.5 uM was studied in vitro on cells of the human mammary tumor cell line HTB123/DU4475 . Selenite and selenocystine affected both cell viability and growth rate of the tumor cells at these selenium concentrations . Selenite and selenocystine decreased intracellular glutathione concentrations, but did not affect tumor cell glutathione peroxidase activity . After six days of exposure to either selenate or selenomethionine, the viability of tumor cells remained stable, but cell growth, as measured by numbers of cells, was retarded . Neither selenate nor selenomethionine produced changes in concentrations of intracellular glutathione . The toxic effect of selenite on tumor cells was enhanced by addition of 0.25 mM glutathione to the growth medium . Preincubation of the tumor cells with 62.5 uM buthionine sulfoximine decreased cellular glutathione to 15% of controls at 24 h and enhanced the toxicity of selenite toward the tumor cells . Glutathione, 2-mercaptoethanol, and L-cysteine were all toxic to the tumor cells in a dose-dependent manner.

J Invest Dermatol, 1991 Aug, 97(2), 364 - 72
Effects of interferons on cultured human melanocytes in vitro: interferon-beta but not-alpha or -gamma inhibit proliferation and all interferons significantly modulate the cell phenotype; Krasagakis K et al.; The effects of human recombinant interferon-alpha-2a (rIFN-alpha), natural interferon-beta (nIFN-beta) and recombinant interferon-gamma (rIFN-gamma) on the proliferation, morphology and antigen expression of cultured human melanocytes were studied in vitro . The investigations were performed in 12-O-tetradecanoylphorbol-13-acetate (TPA)- and serum-containing melanocyte growth medium (MGM), in TPA- and serum-free complete melanocyte medium (CMM) and its mitogen reduced variant (RMM) . In MGM, none of these interferons inhibited the growth of normal melanocytes at concentrations 1-10,000 international units (IU)/ml over a period of 5 d . Only nIFN-beta, dose dependently, inhibited melanocyte proliferation in CMM and RMM in a 6- and 12-d assay (growth inhibition at 10,000 IU/ml; 77-80% of the controls, p less than 0.001) . In contrast, rIFN-alpha and rIFN-gamma exerted no (RMM), or minor effects (CMM) on melanocyte proliferation (only in 12-d assays at 10,000 IU/ml: 24% and 21% of the controls respectively, p less than 0.01) . In parallel experiments performed on melanoma cells, all three interferons were potent inhibitors of proliferation in a 5-d serum-free assay (growth inhibition at 10,000 IU/ml; rIFN-alpha 59%, nIFN-beta 78%, rIFN-gamma 56%, all p less than 0.001) . In addition, nIFN-beta and also rIFN-gamma caused striking morphologic changes of normal melanocytes in vitro . Especially under greater than or equal to 10 IU/ml rIFN-gamma cytoplasmic spreading and flattening of the cultured melanocytes and their nuclei were seen, thus resembling melanoma cells in vitro . Untreated human melanocytes grown in MGM showed high expression of the melanoma-associated antigens HMB-45 (95-100%) and K.1.2 (40-100%), whereas the progression marker A.1.43 was present only on less than 5% of the cells . Cultured melanocytes were 95-100% positive for histocompatibility antigen class I (HLA-I), 30-75% were positive for ICAM-1, whereas they were negative for HLA-DR . After treatment with rIFN-alpha, increased expression of HLA-I antigens was found; nIFN-beta and rIFN-gamma decreased the labeling with HMB-45 (75-100%) and with K.1.2 (25-80%), whereby the expression of A.1.43 was found slightly increased (5-15%) . The HLA class I antigens were upregulated by both nIFN-beta and rIFN-gamma, nIFN-beta being the most potent agent . Also, both nIFN-beta and rIFN-gamma increased the expression of ICAM-1 (nIFN-beta, 75-90%; rIFN-gamma, 90-95%) and induced de novo expression of HLA-DR antigen (nIFN-beta, 15-20%; rIFN-gamma, 65-95%).(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer Commun, 1991 Aug, 3(8), 255 - 64
Involvement of urokinase and its receptor in the invasiveness of human prostatic carcinoma cell lines; Hoosein NM et al.; We have investigated the role of urokinase (UK) and its cell-surface receptor in determining the invasiveness of prostate cancer cells . Three human cell lines, DU-145, PC-3 and LNCaP, that differ in androgen-responsiveness and growth characteristics, were tested . Analysis of the conditioned medium by an enzyme-linked immunosorbent assay showed secretion of UK by DU-145 (63 ng/mL/10(6) cells/48 hr) and PC-3 (682 ng/mL/10(6) cells/48 hr), but absence of secretion by LNCaP cells . Western blot analysis and enzyme activity assay of the conditioned medium confirmed these results . Scatchard analysis of radioligand binding with acid pretreated cells showed the presence of a single population of high affinity UK receptors on DU-145 cells (93,000 sites/cell, Kd = 0.9 nM) and PC-3 cells (25,000 sites/cell, Kd = 1.0 nM) but not on LNCaP cells . DU-145 and PC-3 cells were found to be highly invasive in in vitro invasion assays: 4.5 +/- 0.5% and 6.5 +/- 0.5%, respectively, of total tumor cells (approximately 2 x 10(5)) had penetrated reconstituted basement membrane (Matrigel) in a 72 hr incubation in serum-free growth medium . Under similar conditions, less than 0.25% LNCaP cells invaded Matrigel . The data indicate that androgen unresponsive, aggressive prostate tumor cells of high metastatic potential, DU-145 and PC-3, secrete UK and display cell-surface UK receptors, fully charged with the protease . Conversely, relatively indolent LNCaP cells of low metastatic potential do not secrete UK nor do they possess its binding sites . UK receptor antagonists, UK 12-32 and UK 6-135, which compete with labeled UK for binding to prostatic cells but do not inhibit cellular proliferation or UK secretion, markedly reduced DU-145 and PC-3 cell invasion (80-85% inhibition), thereby suggesting an important role of receptor-bound UK in prostate tumor cell invasion.

Nucleic Acids Res, 1991 Jul 25, 19(14), 3835 - 42
Targeted disruption of a human interferon-inducible gene detected by secretion of human growth hormone; Itzhaki JE et al.; A new method is described for the sib-selection of 'targeted' mammalian cells that have undergone homologous recombination (HR) with a transfected DNA construct . This method has been used to disrupt the 6-16 gene, an interferon (IFN)-inducible gene of unknown function, in two different human cell lines . Disruption was caused by integration of a targeting construct containing a promoterless gene for human growth hormone (hGH) which was expressed after HR with the 6-16 gene . Homologous recombinants were detected in pools of non-homologous recombinants by the appearance of hGH in the growth medium after the addition of IFN . Secondary and tertiary rounds of hGH assays were used to sib-select 9 homologous recombinants that were shown to have 1, 2 or 3 copies of the targeting construct integrated at the 6-16 locus . The method, which should be applicable to other transcribed targets, provides an alternative to selection methods, and offers advantages over other screening methods in being simple, rapid, sensitive and reliable.

FEMS Microbiol Lett, 1991 Jul 15, 66(1), 43 - 7
Osmoregulatory expression of the porin genes in Escherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer; Nakashima K et al.; The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes . The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ . We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs . In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome . It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium . Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T . Mizuno and S . Mizushima, Mol . Microbiol . 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6368 - 71
Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures; Dawson VL et al.; Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices . Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults . We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids . This effect is competitively reversed by L-arginine . Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity . Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation . Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside . These data establish that NO mediates the neurotoxicity of glutamate.

Radiat Res, 1991 Jul, 127(1), 30 - 5
Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids; Reddy NM et al.; Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int . J . Radiat . Biol . 55, 105-117 (1989); Reddy and Lange, Radiat . Res . 119, 338-347 (1989} . Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin . We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum . Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ . Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum . Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids . When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers . The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread . Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum . We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.

J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1743 - 7
The parasitic flagellates Trichomonas vaginalis and Tritrichomonas foetus produce indole and dimethyl disulphide: direct characterization by membrane inlet tandem mass spectrometry; Lloyd D et al.; The use of a membrane inlet triple quadrupole mass spectrometer revealed indole as an end product in the growth medium of cultures of the cattle parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis: formation of indole is enhanced in the presence of added tryptophan . Two different clinical isolates of Trich . vaginalis also produce dimethyl disulphide . Electron impact ionization yielded complex fragmentation mixtures, but the facility for analysis of daughter ions enabled unequivocal assignments . Chemical ionization gave {M + 1}+ species, and tandem mass spectrometry produced identification through daughter ions . The method provides a rapid single-step procedure for the characterization of microbial products without the need for preliminary separation and derivatization.

Zentralbl Veterinarmed B, 1991 Jul, 38(5), 358 - 72
Automation of the resazurin reduction test using fluorometry of microtitration trays; Ali-Vehmas T et al.; Microtitration plate technique and -fluorometry was applied to automate the resazurin reduction test for monitoring bacterial numbers in broth cultures and milk . The effect of resazurin and resorufin concentration, bacterial species, growth medium, pH, and redox potential on the fluorescence response was studied . The timing of the appearance of maximum fluorescence was directly related to the logarithm of the number of colony-forming units (log CFU) . Fresh milk and heat-treated milk contain interfering redox systems . The technique based on microtitration plate fluorometry, when fully automated, seems to provide a high-capacity system for analyzing bacterial numbers in foodstuffs and other media.

J Pak Med Assoc, 1991 Jul, 41(7), 157 - 60
Detection of coliform organisms in drinking water by radiometric method; Khurshid SJ et al.; The radiometric method has been used for detection of coliform bacteria in water . The method is based on measuring the released metabolic 14CO2 from 14C-lactose in growth media containing coliform organisms incubated at 37 degrees C under continuous shaking . This rapid and sensitive radiometric method permits the detection of even single coliform organisms within 6 hours of incubation . Using this automated method, a total of 102 samples (in duplicate) collected from different areas in and around Rawalpindi and Islamabad were assessed for coliform bacteria . Of these 102 samples, 50 were tap water samples, 40 from wells and 6 each were from Rawal and Simly dams . About 47% and 67% tap water samples, while 62% and 74% well water samples were found unsatisfactory from around Islamabad and Rawalpindi areas, respectively . About 83% and 66% water samples from Rawal dam and Simly dam respectively were found to be unsatisfactory.

Toxicol Appl Pharmacol, 1991 Jul, 109(3), 432 - 42
Direct and microsomal activated aflatoxin B1 exposure and its effects on turkey peritoneal macrophage functions in vitro; Neldon-Ortiz DL et al.; Sephadex-elicited turkey peritoneal exudate cells were used to establish adherent macrophage monolayers on glass coverslips in order to determine the effects of aflatoxin B1 (AFB1) on macrophages . Adherent macrophage monolayers were exposed to increasing doses of AFB1 (5, 10, 20, and 40 micrograms), either directly, or to 0.01, 0.1, 0.5, 1, and 5 micrograms of AFB1 in the presence of a chicken microsomal mixed function oxidase system (MFO) . Cultures were incubated with the appropriate treatments for 1 hr, then washed and allowed to recover in fresh growth medium for 2 hr . Direct exposure of macrophages to AFB1 had no detrimental effect on macrophage adherence, percentage damaged, percentage phagocytic, and the number of antibody-coated or uncoated sheep red blood cells internalized per phagocytic macrophage when compared with the sham or solvent treated cultures . However, the addition of MFOs to the cultures treated with much lower doses of AFB1 resulted in significantly higher morphological alterations along with a reduction in cell adherence and phagocytic potential . Addition of piperonyl butoxide (a P450 inhibitor) abrogated AFB1-MFO induced alterations . Data collected in this study suggest that turkey macrophages are resistant to the direct exposure of AFB1 and that AFB1 induced alterations in macrophage effector functions are due to metabolic activation of AFB1 by MFOs.

J Invest Dermatol, 1991 Jul, 97(1), 106 - 10
Autocrine stimulation of interleukin-1 alpha and transforming growth factor alpha production in human keratinocytes and its antagonism by glucocorticoids; Lee SW et al.; Interleukin-1 (IL-1) and transforming growth factor alpha (TGF alpha) mRNA expression was analyzed in cultured normal human keratinocytes . Keratinocytes constititively express IL-1 mRNA when cultured in keratinocyte growth medium but not in Dulbecco's minimal essential medium containing fetal bovine serum, in which the cells differentiate . The predominant form of IL-1 expressed by keratinocytes is IL-1 alpha . Addition of IL-1 alpha to keratinocytes increased IL-1 alpha and TGF alpha mRNA expression in a dose-dependent manner . TGF alpha induced a similar increase in IL-1 alpha and TGF alpha mRNA in keratinocytes . Hydrocortisone decreased the expression of both IL-1 alpha and TGF alpha mRNA in keratinocytes . These findings document an autocrine mechanism by which IL-1 alpha and TGF alpha can stimulate the proliferation of keratinocytes in the skin . It is proposed that this autocrine loop may be hyperactive in psoriasis . Antagonism of the effects of this autocrine loop may be one of the mechanisms by which glucocorticoids exert clinically useful effects in psoriasis and other diseases of the skin.

J Anim Sci, 1991 Jul, 69(7), 3016 - 26
Effects of the inclusion of yeast culture (Saccharomyces cerevisiae plus growth medium) in the diet of dairy cows on milk yield and forage degradation and fermentation patterns in the rumen of steers; Williams PE et al.; The effects of including yeast culture (YC; Saccharomyces cerevisae plus growth medium; 5 x 10(9) organisms/g) in diets for ruminants was examined in two experiments . In Exp . 1, 32 multiparous Friesian dairy cows were fed between wk 7 to 12 of lactation one of four completely mixed diets based on either hay or straw plus rolled barley (mixed to give concentrate:forage ratios of either 50:50 or 60:40, respectively) with or without 10 g YC/d in a 2(3) factorial design . Supplementation with YC increased DM intake of the cows by a mean of 1.2 kg/d (P less than or equal to .062) and increased milk yield by 1.4 liters/d (corrected to 4% butterfat; P less than or equal to .05) . There was an interaction (P less than .05) between diet composition and YC addition; effects of YC were greatest in diets containing 60:40 (concentrate:forage) ratio . In Exp . 2, three steers were fed a diet of 50% hay and 50% rolled barley (DM basis) . Hay was available for the major part of the day but barley was fed in two meals/d . Addition of YC to the diet increased (P less than .05) ruminal pH for 4 h after the barley meal . This elevation in pH probably was due to a reduction (P less than or equal to .01) in the concentration of L-lactate in the ruminal liquor of steers given YC (1.43 vs 3.55 mM; P less than or equal to .01) . Peak ruminal L-lactate concentration (7.75 mM) in the controls coincided with time of minimum pH values (2 h after the meal of barley); this peak was absent in steers given YC . YC had no effect on the concentration of VFA in ruminal liquor, but the ratio of acetate to propionate was reduced (P less than or equal to .01) from 3.3:1 to 2.8:1 in steers given YC . The extent of DM degradation of hay incubated in the rumen of steers fed the hay and rolled barley diet was increased (P less than .05) in the presence of YC at 12 h of incubation, but degradation was similar in all treatment groups after 24 h of incubation . Presence of yeast culture in the rumen had effects on ruminal stoichiometry . An increased rate of forage degradation may have increased forage intake and productivity of these dairy cows.

Mol Microbiol, 1991 Jul, 5(7), 1745 - 53
The mdoA locus of Escherichia coli consists of an operon under osmotic control; Lacroix JM et al.; In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane-derived oligosaccharides (MDOs) . The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene-fusion analysis . A 'neo' cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid . This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene . Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter . Moreover, the 'neo' cassette inactivated another, uncharacterized, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished . The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA . Two groups were defined, and the two genes are organized into an operon (mdoGH) . This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene . An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ . The hybrid beta-galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis . This regulation was unaffected by the presence, or absence, of MDOs in the periplasm . Finally, the amount of mdoA-specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.

Hum Antibodies Hybridomas, 1991 Jul, 2(3), 155 - 9
Parameters to enhance human hybridoma formation with hypoosmolar electrofusion; Perkins S et al.; Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 10(5) B cells . A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus--activated B cells and mouse-human heteromyelomas . This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells . The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B cells . The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer . By manipulating these parameters, a high hybrid yield can be achieved with different mouse-human heteromyelomas and Epstein-Barr virus-activated B cells.

J Biol Chem, 1991 Jun 25, 266(18), 11753 - 60
Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage . Role of guanosine tetraphosphate and ribosome feedback; Baracchini E et al.; The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis . Experiments were carried out in different growth media and with two different strains of E . coli, B/r and K-12 . The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt . 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain . 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes . 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes . An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.

Cancer Res, 1991 Jun 15, 51(12), 3304 - 10
Regulation of growth by a nerve growth factor-like protein which modulates paracrine interactions between a neoplastic epithelial cell line and stromal cells of the human prostate; Djakiew D et al.; Nerve growth factor-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite outgrowth of PC12 cells . Nerve growth factor-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells . Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody . NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions . The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite outgrowth from PC12 cells . This neurite outgrowth activity was partially inhibited by treatment with NGF antibody . Neither the serum containing growth medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite outgrowth . The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic growth between TSU-pr1 cells and HPS cells . The relative growth of TSU-pr1 cells, as indicated by {3H}thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner . In the converse interaction, the relative growth of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose-dependent manner . Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic fibroblast growth factor, and basic fibroblast growth factor . Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (20 micrograms/ml) also reduced TSU-pr1 proliferation to 16% of maximal levels . Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic fibroblast growth factor and basic fibroblast growth factor did not inhibit the proliferation of either cell type . These results suggest that TSU-pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive growth in vitro.

Cancer Res, 1991 Jun 15, 51(12), 3204 - 11
Additive and supraadditive interaction between ionizing radiation and pazelliptine, a DNA topoisomerase inhibitor, in Chinese hamster V-79 fibroblasts; Balosso J et al.; The cytotoxic effect of the 9-azaellipticine derivative pazelliptine in combination with gamma-ray irradiation was investigated using Chinese hamster V-79 cells in culture . gamma-ray irradiation and drug treatment (1-h drug exposure) were applied at 1-h intervals for partial DNA damage recovery in growth medium . Isobologram analysis of the clonogenic potential gave evidence of supraadditive interaction in the radiation----drug sequence with 10% survival as an endpoint . No synergistic potentiation was observed at higher survival or as pazelliptine was applied first . Pazelliptine abolished the low-dose shoulder characteristic of asynchronous cell response to gamma-rays . Although rejoining of radiation-induced DNA strand breaks was completed at the time of drug exposure, pazelliptine brought about a larger amount of DNA strand breaks in preirradiated than in nonirradiated cells . The time and dose dependencies of DNA strand break formation and repair with radiation and/or pazelliptine were analyzed by neutral and alkaline filter elution . Pazelliptine in the micromolar range showed the same pattern of double-stranded cleavable complex formation as expected of a DNA topoisomerase II-targeting agent . At a low concentration of pazelliptine, however, protein-concealed breaks were mostly in the form of single-stranded adducts . Such single-stranded complexes have been reported to occur with some topoisomerase II-targeting drugs; their properties are also reminiscent of those induced by the topoisomerase I poison, camptothecin . It is proposed that topoisomerase poisoning interacts with the repair of radiation-induced lesions.

Mol Gen Genet, 1991 Jun, 227(2), 238 - 44
Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism; Harder PA et al.; Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2 . Mutants of S . griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas . Genetic evidence indicated that this phenotype was the result of a deletion of greater than 15 kb of DNA, including the structural genes for cytochrome P-450SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively) . In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the subC,B gene products (cytochrome P-450SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea . These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450SU2 in the absence of P-450SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S . griseolus.

Antimicrob Agents Chemother, 1991 Jun, 35(6), 1116 - 26
Synthesis and antimicrobial evaluation of dirithromycin (AS-E 136; LY237216), a new macrolide antibiotic derived from erythromycin; Counter FT et al.; Dirithromycin is a 9-N-11-O-oxazine derivative which is formed by condensation of 9(S)-erythromycylamine with 2-(2-methoxyethoxy)acetaldehyde . Dirithromycin is hydrolyzed, either under acidic conditions or in vivo, to its major active metabolite, 9(S)-erythromycylamine . The antimicrobial spectrum of dirithromycin is similar to that of erythromycin; both antibiotics are active against gram-positive bacteria, Legionella spp., Helicobacter pylori, and Chlamydia trachomatis . Comparable results were obtained for each antibiotic in MIC and MBC determinations and in the potential development of resistance in vitro . The effects of human serum, bacterial growth media, test methodology, and inoculum size on MICs were similar for each antibiotic . In standard mouse protection studies, dirithromycin was more efficacious than erythromycin against experimental infections after subcutaneous administration of antibiotic . These results were consistent with pharmacokinetic studies in rodents, which showed that dirithromycin gave more persistent concentrations of antibiotic in serum and tissues than were achieved with erythromycin . These studies indicate that dirithromycin possesses antimicrobial activity comparable to that of erythromycin in vitro but is more active than erythromycin in vivo, which may be attributable to the persistence of antimicrobial activity in the tissue(s) of the test animals.

Curr Genet, 1991 Jun, 19(6), 423 - 7
Hyper-resistance to nitrogen mustard in Saccharomyces cerevisiae is caused by defective choline transport; Li ZY et al.; The recessive hnm1 mutant allele is responsible for hyper-resistance to nitrogen mustard in Saccharomyces cerevisiae . Transformation with a single-copy HNM1 wild-type allele of such hyper-resistant mutants will restore wild-type sensitivity to nitrogen mustard . By contrast the presence of multi-copy vectors containing HNM1, in either a hyper-resistant hnm1 mutant or an HNM1 wild-type, will lead to a novel, mustard-sensitive phenotype unrelated to defects in DNA repair genes . Gene disruption of HNM1 revealed that this gene is non-essential for cells prototrophic for choline (CHO1) but lethal for cells with a cho1 genotype . Sensitivity to nitrogen mustard of wild-type HNM1, but not of hnm1 mutants, depends on the choline content of the growth medium, with cells grown in choline-free medium exhibiting the highest sensitivity . Sequencing of a 300 bp DNA fragment of HNM1 revealed the identity of this gene with the CTR locus, which is responsible for choline transport in Saccharomyces cerevisiae.

Ecotoxicol Environ Saf, 1991 Jun, 21(3), 235 - 9
Analysis of organotin uptake in Escherichia coli K-12; Singh K et al.; The effect of tributyltin chloride (TBT) was potentiated by the presence of Tris ions in the growth medium compared with phosphate ions . The growth of Escherichia coli in the presence of Tris ions was inhibited completely at a concentration of 25 microM TBT whereas 79% growth was recorded in the presence of phosphate ions . Tris ions caused more protein to be released into the medium than phosphate ions . However, apart from protein release the rate of organotin uptake was less in Tris than in phosphate ions . Phosphate ions may play a positive role in the active accumulation of organotin into E . coli cells by providing cellular energy.

Cryobiology, 1991 Jun, 28(3), 246 - 50
Cryosurvival of Trichomonas vaginalis during cryopreservation of human semen; Sherman JK et al.; Despite a 90% cryosurvival of Trichomonas vaginalis in their growth medium trypticase yeast maltose (TYM) with DMSO, none of these parasites have previously been observed to survive during cryopreservation of infected human semen with glycerol (Andrologia 18, 323 (1986)) . This could have been due to the failure of the culture method used to detect low numbers of survivors . The prospects of possible transmission of T . vaginalis by artificial insemination with cryobanked (-196 degrees C) semen prompted an investigation of the cryosurvival of this parasite in the presence of semen with the cryoprotectant glycerol, using a more sensitive culture method for viability evaluation . Semen and seminal fluid from the same 23 ejaculates, as well as culture medium, were inoculated with small clinical numbers of T . vaginalis and evaluated as to their survival before and after cryopreservation . Results indicated: (i) The highest cryosurvival of T . vaginalis (4.5%) was in cryobanked (glycerolated) semen, (ii) semen, as well as glycerol, shows cryoprotective action, and (iii) glycerol reduced survival of parasites in semen, seminal fluid, and TYM medium during exposure prior to freezing . Clinical information on infectivity of small numbers of T . vaginalis and the data presented here suggests that these organisms could be transmitted by artificial insemination with infected cryobanked human semen.

J Vet Med Sci, 1991 Jun, 53(3), 439 - 46
Establishment and characterization of a thymidine kinase deficient avian fibroblast cell line derived from a Japanese quail cell line, QT35; Niikura M et al.; An avian thymidine kinase deficient (TK-) fibroblast cell line (QTTK-) was established from a Japanese quail cell line, QT35, and characterized the biological properties . QTTK- could grow in the presence of 5-bromo-2'-deoxyuridine (BUdR, 100 micrograms/ml) and not in the growth medium with hypoxanthine-aminopterin-thymidine . Compared to QT35 cells, the 3H-thymidine incorporation of the QTTK- cells and the TK activity of the cell extract significantly decreased to 0.3% and 0.5%, respectively . In the thymidylate synthetase activity, the karyotype, and the sensitivities to either fowlpox virus (FPV) or herpesvirus of turkeys (HVT), OTTK- cells were similar to the parental QT35 cells . Since QTTK- cells were permissive to FPV and HVT infections and these viruses could not grow in the presence of 50 to 75 micrograms/ml of BUdR, QTTK- cells may be useful for the construction of recombinant FPV and HVT that have foreign genes within the TK gene of the virus genome.

J Cell Sci, 1991 Jun, 99 ( Pt 2), 397 - 405
Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes; Watt FM et al.; During terminal differentiation, keratinocytes lose the ability to divide . One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ({Ca2+}i) . The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in {Ca2+}i in response to factors that stimulate {3H}thymidine incorporation and increase {Ca2+}i in undifferentiated keratinocytes . {Ca2+}i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method . Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation . Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM) . Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased {3H}thymidine incorporation . A switch from low calcium to standard medium also stimulated {3H}thymidine incorporation whether or not the cells had been serum-starved . In each experiment some cells showed an increase in {Ca2+}i while others did not . However, the heterogeneity in the {Ca2+}i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations . Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in {Ca2+}i in response to serum-containing medium . Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in {Ca2+}i.(ABSTRACT TRUNCATED AT 250 WORDS)

J Allergy Clin Immunol, 1991 Jun, 87(6), 1035 - 42
House dust mite-derived amylase: allergenicity and physicochemical characterization; Lake FR et al.; Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium . It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm) . Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01) . Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing . It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix . The optimum pH for enzymatic activity was revealed to be 6.4 . IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds . Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase . IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay.

J Bacteriol, 1991 Jun, 173(11), 3523 - 30
A novel transcriptional response by the cat gene during slow growth of Escherichia coli; Meyer BJ et al.; A novel response to growth rate was found with expression of the chloramphenicol acetyltransferase (cat) gene in Escherichia coli . The amount of cat mRNA relative to total RNA increased about 11-fold as growth rates decreased 5- to 6-fold, without an increase in translation . The accumulation of cat mRNA was in contrast to decreased cellular concentrations of total RNA, trxA, ompA, or 23S rRNA as the growth rate decreased and was not due to changes in gene dosage or mRNA stability . Stability of the cat mRNA does not appear to be regulated by growth rate . No significant change in either chemical or functional stability was observed within a five- to sixfold range of growth rates when chemostat-grown cells were used . However, cat mRNA stability was affected by growth medium composition . The half-life of cat mRNA decreased about threefold, with an approximate fourfold increase in generation time due to changes in growth medium . Transcriptional studies have indicated that accumulation of cat mRNA at slow growth rates is the result of a specific transcriptional response to changes in cellular generation times . We propose that increases in the cellular concentration of a specific message at slow growth rates may reflect an additional type of survival response in E . coli.

Eur J Biochem, 1991 Jun 1, 198(2), 365 - 73
N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase; Reinbothe S et al.; Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide . Two different mechanisms of tolerance to the herbicide were observed . One is characterized by the overproduction and 40-fold accumulation of the target enzyme . 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine . The other is connected with a herbicide-insensitive enzyme . No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena . Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide . Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein . Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system . In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro . In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethyl)glycine.

J Bacteriol, 1991 Jun, 173(12), 3749 - 55
Intramolecular second-site revertants to the phosphorylation site mutation in OmpR, a kinase-dependent transcriptional activator in Escherichia coli; Brissette RE et al.; OmpR is a transcriptional activator for the ompF and ompC genes of Escherichia coli . Its phosphorylation is mediated by a transmembrane sensory-receptor protein, EnvZ, and is essential for transcriptional activation . In a previous study, when the aspartic acid residue at position 55, the putative phosphorylation site, was replaced with glutamine (D55Q), ompF and ompC expression were completely lost . In this study two pseudorevertants of the D55Q mutation were isolated and identified to be the replacement of threonine at position 83 with alanine (T83A) and glycine at position 94 with serine (G94S) . The revertant OmpRs no longer responded to EnvZ function when ompF and ompC expression were examined . The purified D55Q-T83A OmpR was unable to be phosphorylated by EnvZ in vitro . The role of EnvZ as an osmosensor for the environmentally regulated expression of OmpF and OmpC has been indicated in previous studies . The isolation of seemingly EnvZ-independent OmpR revertants in this study, however, made it possible to examine the osmolarity-regulated expression of OmpF and OmpC in the absence of effects exerted by EnvZ . We found that the expression of OmpF and OmpC supported by these revertant OmpRs was clearly regulated in accordance with the change in osmolarity of the growth media . These results indicate that another EnvZ-independent mechanism(s) may also contribute to the regulated expression of the ompF and ompC genes.

Int J Neurosci, 1991 Jun, 58(3-4), 249 - 54
Muscle derived motoneuron trophic factors promote the survival of motoneurons in vitro only when serum is present in the growth medium; Juurlink BH et al.; Motoneuron cultures were established from E6 chick spinal cord . Motoneurons survived for less than 2 days in chemically defined medium . The addition of muscle extract to the medium supported the survival of only a small portion (approximately 2%) of motoneurons for 8 days in vitro . A similar low survival rate was observed when the growth medium was supplemented with serum . The addition of muscle extract to serum containing medium resulted in the survival of about 20% of the motoneurons for 8 days . No differences were seen in the ability of tissue extracts prepared from E8 hindlimb, or muscle obtained from E11, E15, E18 and P3 chicks to support motoneuron survival in the presence of serum . It is apparent that although there are trophic factors present in muscle that support motoneuron survival in vitro, the actions of such trophic factors are dependent upon the presence of yet other factors found in serum.

Brain Res, 1991 May 31, 550(1), 69 - 76
Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain; Wiesinger H et al.; Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells . When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria . The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells . Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency . Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.

FEMS Microbiol Lett, 1991 May 15, 64(2-3), 189 - 94
Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis; Burra SS et al.; When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed . Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above) . Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium . Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM . Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium . Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.

Biochemistry, 1991 May 7, 30(18), 4491 - 4
Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II; Venters RA et al.; Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of {13C6, 99%}glucose . We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium {1,2-13C2, 99%}acetate as the sole carbon source and {15N, 99%}ammonium chloride as the sole nitrogen source . In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium {1-13C, 99%}acetate and sodium {2-13C, 99%}acetate in conjunction with unlabeled sodium acetate . This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates . These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II . The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium {1,2-13C2, 99%}acetate growth media is considerably less than the cost of the {13C6, 99%}glucose growth media . We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

Jpn J Cancer Res, 1991 May, 82(5), 493 - 6
Serum requirement for in vitro invasion by tumor cells; Imamura F et al.; The effect of fetal calf serum (FCS) on in vitro invasion by rat ascites hepatoma cells (AH130) was studied by using the in vitro invasion assay . Although the coculture of the highly invasive clone (MM1) of AH130 cells and the mesothelial cell layer or endothelial cell layer in modified minimum essential medium supplemented with 10% FCS resulted in extensive penetration of the layer by the tumor cells, the omission of FCS resulted in an almost complete elimination of the in vitro invasion . The in vitro invasiveness by human small cell lung cancer cells (OC10) was also remarkably reduced by the omission of FCS from the assay medium, suggesting a requirement of serum for the in vitro tumor cell invasion . When 10% FCS was added to the medium 2 h after the tumor cell seeding in FCS-free invasion assay system, penetration by MM1 cells was observed within an hour . This rate of penetration was almost the same as that when 10% FCS was added at the time of tumor cell seeding . FCS was also required for the penetration of a mesothelial cell monolayer by MM1 cells in a defined growth medium (SFM-101), in which MM1 cells were well maintained . The invasion-inducing activity appears to be independent of the growth-stimulating activity in serum.

Can J Microbiol, 1991 May, 37(5), 361 - 7
Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum; Park WS et al.; The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication . The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity . Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity . High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source) . Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient . Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria . Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate . Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase . Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose . Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes . Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.

J Gen Microbiol, 1991 May, 137 ( Pt 5), 1087 - 92
Isolation and purification of viable Ureaplasma urealyticum cells free from medium components; Horowitz S et al.; A procedure was devised to produce Ureaplasma urealyticum preparations free of adsorbed components of the growth medium, which contains high concentrations of serum . The ureaplasmas were cultivated in a medium containing PPLO-serum fraction as a replacement for horse serum . High titres of ureaplasmas (greater than 10(7) c.f.u . ml-1) were obtained . Harvested cells were then purified by Urografin density gradient centrifugation . By use of 3H-labelled ureaplasma cells and 125I-labelled medium components, a distinct band of viable cells devoid of serum constituents was demonstrated . The absence of medium components was verified by immunoblotting cells from this band with antiserum to medium components . Medium components that had been present before the purification procedure were undetectable in the purified cell fraction obtained . The viability of the purified ureaplasma cells represented an 85% recovery rate and their antigenicity, examined with anti-serotype specific antiserum, remained intact . This easy and reproducible procedure can be used to prepare purified ureaplasmas for investigation of ureaplasmal antigens and their expression and/or role in disease.

J Gen Microbiol, 1991 May, 137 ( Pt 5), 1081 - 6
Tubular spinae are long-distance connectors between bacteria; Bayer ME et al.; The marine pseudomonad D71 (NCMB 2018) {'Spinomonas maritima'} can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity . In general, spina-carrying cells show these appendages with open distal ends . We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection . By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers . Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer . The inner and outer cell membranes were often joined at spina-insertion areas . Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns . We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.

Res Microbiol, 1991 May, 142(4), 359 - 71
The repression of trehalose transport and metabolism in Escherichia coli by high osmolarity is mediated by trehalose-6-phosphate phosphatase; Klein W et al.; Trehalose transport and metabolism in Escherichia coli are induced by trehalose in the growth medium but only at low osmolarity . In contrast, synthesis of internal trehalose as an osmoprotectant occurs only at high osmolarity, independent of the carbon source . The synthesis of internal trehalose proceeds via the UDP-glucose-mediated transfer of glucose to glucose-6-phosphate, forming trehalose-6-phosphate, which is then hydrolysed to trehalose . We demonstrate that the inducer for the synthesis of the trehalose transport system as well as of amylotrehalase, the key enzyme in trehalose metabolism at low osmolarity, is trehalose-6-phosphate . We found that the inability to induce these proteins at high osmolarity is primarily due to activity of trehalose-6-phosphate phosphatase, the enzyme responsible for the final step in the synthesis of internal trehalose under these conditions . A gene, otsP, necessary for the synthesis of the biosynthetic trehalose-6-phosphate phosphatase, is located at min 42 closely linked to otsA/B the structural genes for the trehalose-6-phosphate synthase . There is another gene locus near 84 min on the chromosome, that we termed otsR, which is involved in the regulation of otsA/B and possibly otsP . The nature of this regulatory gene is unclear at present.

Cancer Res, 1991 Apr 15, 51(8), 2179 - 84
Quantitation, in vitro propagation, and characterization of preleukemic cells induced by radiation leukemia virus; Yefenof E et al.; Intrathymic (i.t.) inoculation of radiation leukemia virus into C57BL/6 mice induces a population of preleukemic (PL) cells that can progress into mature thymic lymphomas upon transfer into syngeneic recipients . A minimum of 10(3) PL thymic cells are required to induce lymphomas in the recipient . Most of the individual lymphomas developed in mice which were inoculated with cells of a single PL thymus, derived from different T-cell precursors . PL thymic cells could be grown in vitro on a feeder layer consisting of splenic stromal cells . Growth medium was supplemented with supernatant harvested from an established radiation leukemia virus-induced lymphoma cell line (SR4) . The in vitro-grown PL cells were characterized as Thy-1+, CD4+, CD8- T-cells, most of which expressed radiation leukemia virus antigens . Cultured PL cells were found to be nontumorigenic, based on their inability to form s.c . tumors . However, these cells could develop into thymic lymphomas if inoculated i.t . into syngeneic recipients . A culture of PL cells, maintained for 2 mo, showed clonal T-cell receptor arrangement . Lymphomas which developed in several recipient mice upon injection with these PL cells were found to possess the same T-cell receptor arrangement . These results indicate that PL cells can be adapted for in vitro growth while maintaining their preleukemic character.

J Appl Bacteriol, 1991 Apr, 70(4), 351 - 4
Neutralization efficacy of Dey-Engley medium in testing of contact lens disinfecting solutions; Sutton SV et al.; A quantitative assay for the demonstration of neutralizer efficacy was developed to monitor contact lens disinfecting solutions . Adequate neutralization of disinfecting agents is essential to the accurate determination of disinfecting activity with time . This method employed the recovery of small numbers of micro-organisms from neutralizing medium containing a disinfectant . A statistical estimation of significance between treatments demonstrated that Dey-Engley medium (DE; Difco) was generally effective when tested as an agar growth medium with several bacterial test organisms . DE medium from another vendor was less effective, underscoring the need for laboratory quality control and monitoring . DE agar (Difco) adequately neutralized all solutions tested at a 1:20 dilution . The solutions included those containing Dymed (polyaminopropyl biguanide, 0.00005%), chlorhexidine (0.005%), Polyquad (0.001%), chlorhexidine (0.005%) and thimerosal (BP, 0.001%), thimerosal (BP, 0.002%) and Tris(2-hydroxyethyl) tallow ammonium chloride (0.013%), and a solution preserved with 115 ppm benzalkonium chloride (BAK) . A modification of this medium was developed which retained virtually all of the neutralizing efficacy for the solutions tested while allowing the use of automated testing procedures.

Exp Eye Res, 1991 Apr, 52(4), 461 - 4
RPE cells from normal rats do not secrete a factor which enhances the phagocytosis of ROS by dystrophic rat RPE cells; Hall MO et al.; Retinal pigment epithelial (RPE) cells from normal and dystrophic rats were grown separately and in mixed culture for 7 days, without a change of growth medium . Isolated rod outer segments (ROS) were suspended in the conditioned medium from these cells, and were fed to the mixed or pure RPE cell cultures . No increase or decrease in the phagocytosis of ROS by dystrophic or normal RPE cells, respectively, was observed . These results suggest that normal RPE cells do not secrete a diffusible factor(s) which enhances the phagocytosis of ROS by dystrophic RPE cells.

Can J Microbiol, 1991 Apr, 37(4), 317 - 21
Calcium binds to and is required for biological activity of the 104-kilodalton hemolysin produced by Actinobacillus pleuropneumoniae serotype 1; Devenish J et al.; Actinobacillus pleuropneumoniae serotype 1, strain Shope 4074, was grown on agar medium containing 10 mM Ca2+ and under Ca2+ limiting conditions by addition of 2 and 5 mM EGTA to the growth medium . Hemolysis of washed bovine erythrocytes was observed from the culture grown in Ca2+ excess but not from the two cultures where Ca2+ was chelated from the growth medium by using EGTA . However, the hemolytic activity of these latter two cultures could be restored if 10 mM Ca2+ was added to the dilution buffer . This restored hemolytic activity could be neutralized with a rabbit homologous polyclonal antiserum specific for the 104-kDa hemolysin of serotype 1 but not with preimmune serum from the same rabbit . Stained SDS-PAGE gels and immunoblots showed the 104-kDa protein hemolysin in fractions from each of the three growth conditions . Thus, the restored hemolytic activity was associated with the 104-kDa protein in the three cultures . In addition, the 104-kDa protein, when electroblotted onto nylon membranes, bound 45Ca, indicating that the molecule has binding sites for Ca2+ . The results indicate that Ca2+ is required for the biological activity of the 104-kDa hemolysin of A . pleuropneumoniae serotype 1.

Arch Biochem Biophys, 1991 Apr, 286(1), 85 - 93
Modulation of fibroblast motility by a cytosolic extract of Cyanobacteria; Hodara ML et al.; Proliferation and migratory behavior of L929 murine fibroblasts were shown to be modified in the presence of a cytosolic extract of Phormidium sp . (Cyanobacteria) . The addition of Phormidium extract to the growth medium (Dulbecco's modified Eagle's medium) supplemented with 0.5% newborn calf serum increased cell proliferation . The effect was shown to be cell line specific . A quantitative analysis performed according to De Laat, Tertoolen, and Bluemink (1981, Eur . J . Cell Biol., 23, 273-279), showed that Phormidium extract was a potential aggregative effector for fibroblasts . Heating (100 degrees C, 4 min) inactivated the clustering effect of the extract, but the effect on cell proliferation was retained . A video analysis of cells after divisions showed that the extract activated cell migration in the same way as 5% serum did during the first 24 h of treatment . Between 24 and 48 h of treatment, cell migration in the presence of the extract was inhibited when compared to migration in 0.5 or 5% serum . We have shown that Phormidium extract may contain two or three kinds of effectors which acted as exogenous growth factors (allowing attachment and proliferation) and as modulator(s) of the cell migratory behavior (activator of migration in early times of the growth and inhibitor later).

Nutr Clin Pract, 1991 Apr, 6(2), 49 - 54
Infection control of parenteral nutrition solutions; Thompson B et al.; Microbial contamination of parenteral nutrition solutions is a preventable cause of in patients receiving nutrition support . The components of the parenteral nutrition solutions have variable microbial growth potential . Crystalline amino acid and dextrose solutions are poor growth media for bacteria but may support fungal growth . Lipid emulsions provide an excellent medium for growth of bacteria and fungal species . Total nutrient admixtures will support microbial growth better than standard parenteral nutrition solutions will but less well than will lipid emulsion alone . Control of infection related to contaminated infusate depends on compounding procedure, quality control, appropriate storage, and procedures to prevent in-use contamination . Guidelines are presented for the preparation and administration of parenteral nutrition infusates that will minimize microbial contamination.

In Vitro Cell Dev Biol, 1991 Apr, 27A(4), 312 - 26
A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells; Gordon EL et al.; A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated . RCMECs required fetal bovine serum to attach efficiently . Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium . Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes . These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure . By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures . RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements . RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures . The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture . RCMECs had higher enzymatic activity than RAECs . In both RCMECs and RAECs enzyme activity decreased with time in culture . The function of endothelial cells is specialized depending on its location . This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization . These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.

Endod Dent Traumatol, 1991 Apr, 7(2), 69 - 72
Vitality of human lip fibroblasts in milk, Hanks balanced salt solution and Viaspan storage media; Hiltz J et al.; The purpose of the study was to compare the effect of storage in milk, Hanks balanced salt solution (HBSS) and Viaspan on the vitality of human lip fibroblasts . Confluent monolayers of the fibroblasts were grown in petri dishes . The growth medium was poured off and replaced in 27 dishes each with Viaspan, fresh whole milk and Hanks balanced salt solution respectively . Three of the original plates were analyzed at time zero to obtain the average number of cells each plate contained at confluence . At times ranging from 2 to 168 hours the average number of vital cells remaining was measured using the trypan blue exclusion test . The groups stored in milk maintained a high percentage of vital cells for 6 hours (68.2%) . At 12 hours milk's effectiveness had dropped to 43.4% vital cells and it was not effective at all at 48 hours (0.024% vital cells) . H.B.S.S . was extremely effective for 24 hours with 71.3% vital cells remaining . At 48 hours, the percentage of vital cells dropped to 38.0% and by 120 hours no cells survived . Viaspan was the most effective storage medium at all observation periods and at 168 hours still had 37.6% vital cells present.

Protein Eng, 1991 Apr, 4(4), 485 - 92
Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo; Jespers L et al.; The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids . Two methods for improving the efficiency of translation have been tested . First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS . The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS . The expression levels are described by an approximately Gaussian histogram . The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor . Under the expression conditions, the protein is partially proteolysed . The proteolysis is significantly decreased by adding salt to the growth medium . After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions . An efficient purification protocol is described.

J Gen Microbiol, 1991 Apr, 137 ( Pt 4), 745 - 50
The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture; Welsh DT et al.; Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl . Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant . The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis . Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic . The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption . Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.

Cell Growth Differ, 1991 Apr, 2(4), 209 - 14
Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells; Woessner RD et al.; The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells . When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis . The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle . When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0 . In contrast, the 180kD enzyme was still present after all of the cells had entered G0 . The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei . The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme . The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.

Biochem Biophys Res Commun, 1991 Mar 15, 175(2), 706 - 12
The amino-terminal region of a proteochondroitin core protein, secreted by aortic smooth muscle cells, shares sequence homology with the pre-propeptide region of the biglycan core protein from human bone; Marcum JA et al.; Smooth muscle cells, isolated from rat and bovine aortae and grown in vitro, synthesize chondroitin sulfate proteoglycans which are secreted into the growth media . Analysis of metabolically {35S}-labeled macromolecules, employing ion-exchange chromatography, revealed a single peak of radioactivity, upon elution with a linear salt gradient . Treatment of the material with enzymes that specifically degrade chondroitin sulfate demonstrated that chondroitin-4-sulfate was the predominant species isolated from rat smooth muscle cells and that chondroitin-4-sulfate and dermatan sulfate were the predominant species isolated from bovine aortic smooth muscle cells . Treatment of the native proteoglycans with chondroitinase ABC and subsequent SDS-PAGE analysis of the digestion products resulted in the appearance of a band with an apparent molecular weight of 45,000 . Electrotransfer of the core protein to Immobilon-P membrane and gas phase sequencing of the amino-terminal region revealed striking homology between the core proteins of the rat and bovine proteochondroitin with the pre-propeptide region of human bone biglycan.

Cancer Res, 1991 Mar 15, 51(6), 1600 - 5
8-Chloroadenosine 3',5'-monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite; Van Lookeren Campagne MM et al.; 8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations . We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells . Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate . Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.

Antimicrob Agents Chemother, 1991 Mar, 35(3), 477 - 83
Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility; Malouin F et al.; Cell composition and surface properties of Escherichia coli were modified by using various growth media to investigate the role of yet uncharacterized components in ceftazidime susceptibility . An eightfold dilution of Luria broth was used as the basic growth medium and was supplemented with up to 4% phosphate, 5% glucose, or 12% L-glutamate . Decreases in cephaloridine and ceftazidime susceptibility, of two- and eightfold, respectively, were observed only in the glucose-enriched medium . The outer membrane permeability to ceftazidime and cephaloridine was evaluated by crypticity indices . Indices were unchanged under all growth conditions . Fluorometry of whole cells with 1-N-phenylnaphthylamine showed that glucose does not affect the interaction of this hydrophobic probe with the membranes but showed that elevated concentrations of phosphate or glutamate cause a marked increase in cell hydrophobicity, which, in turn, correlates with an increase in the susceptibility of E . coli to nalidixic acid . Growth in phosphate- or glutamate-enriched media caused an augmentation in major phospholipid species and may explain the increased hydrophobicity and susceptibility of E . coli to nalidixic acid . These data showed that E . coli susceptibility to ceftazidime is not influenced by cell surface hydrophobicity and suggested that the contribution of a nonspecific lipophilic diffusion route for entry of ceftazidime into cells is not likely to occur or is distinct from that of more hydrophobic molecules such as nalidixic acid . Finally, the penicillin-binding proteins of the E . coli cells were also investigated . Penicillin-binding protein 8 was only markedly labeled with 125I-penicillin V in inner membranes extracted from cells grown with glucose . Results of this study suggest that the unexpected change in penicillin-binding protein 8 observed in the presence of glucose may be responsible for the increase in MICs of cephaloridine and ceftazidime.

Brain Res Bull, 1991 Mar, 26(3), 429 - 32
Abnormalities in the spontaneous firing patterns of cultured rat neocortical neurons after chronic exposure to picrotoxin during development in vitro; Ramakers GJ et al.; We have used the GABA-A antagonist picrotoxin (PTX) to investigate whether chronic disinhibition, leading to intensified neuronal firing, would induce a specific pattern of physiological alterations in cultured rat neocortex cells . Overall mean spontaneous discharge rates were little affected by 1 microM PTX but firing occurred mainly as repetitive high-frequency bursts of action potentials . This "phasic" pattern contrasted with the irregular, quasi-random, firing usually seen in control units . Neurons tested in normal growth medium after prolonged exposure to 1 microM PTX showed weaker interspike interval dependencies (Markov value) than in controls, along with reduced regularity in the occurrence of bursts . Since all physiological changes were opposite in direction to those reported earlier after chronic suppression of bioelectric activity, the results support the hypothesis that endogenous synaptic and/or action potentials are important for the maturation of neocortical networks . Since experimental alterations were found only in spike-train parameters which reflect ontogenetic changes in untreated control cultures, GABAergic inhibition (by preventing neuronal discharges from becoming too intense) presumably serves to constrain the rate of development within optimal limits.

J Bacteriol, 1991 Mar, 173(6), 2006 - 10
A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase; Payton MA et al.; A temperature-sensitive mutant of Saccharomyces cerevisiae was identified which at the restrictive temperature of 37 degrees C is unable to secrete a number of cell wall-associated proteins and thus resembles previously reported sec mutants . In contrast to other sec mutants, however, both the temperature-sensitive growth and the secretion defects can be repaired by the addition of D-mannose to growth media . We show that the mutant possesses a single, apparently recessive mutation which leads to the production of a thermolabile phosphomannose isomerase.

EMBO J, 1991 Mar, 10(3), 555 - 62
Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival; Heinemeyer W et al.; Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells . We have isolated three mutants affecting the proteolytic activity of proteinase yscE . The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2 . The PRE1 gene was cloned and shown to be essential . The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms . Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits . Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome . Diploids homozygous for pre1-1 are defective in sporulation . Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature . Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.

Radiat Res, 1991 Mar, 125(3), 267 - 76
Depletion of glutathione after gamma irradiation modifies survival; Saunders EL et al.; The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells . Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM) . Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting . Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95% . Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures . Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation . The DEM/BSO exposure did not affect D0 . To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures . Levels of GSH were depleted by 75% by this protocol . Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM . Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75% . In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM . Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM . In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h . This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO . Survival was not affected if the cells were exposed to the DEM or BSO alone . This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained . The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure . These experimental results in addition to previous ones (Freeman and Meredith, Int . J . Radiat . Oncol . Biol . Phys . 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.

Arch Biochem Biophys, 1991 Mar, 285(2), 297 - 305
Calcium modulation of polyamine transport is lost in a putrescine-sensitive mutant of Neurospora crassa; Davis RH et al.; Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane . We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+ . The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid . The mutant phenotype also appears in the wild type after treatment with cycloheximide . The results suggest that putrescine uptake is normally regulated by an unstable Ca(2+)-binding protein that restricts polyamine uptake . This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells . The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-1+ gene . Preliminary evidence suggests that the putrescine carrier is not the Ca(2+)-sensitive, low-affinity K(+)-transport system, but K+ efflux does accompany putrescine uptake.

Appl Environ Microbiol, 1991 Mar, 57(3), 830 - 5
In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae; Rosa MF et al.; Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% {vol/vol}), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae . The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol) . The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical . A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol . The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation . After ethanol removal, the rapid in vivo reversion of ATPase activation was observed . While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro . The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.

Cytotechnology, 1991 Mar, 5(3), 273 - 7
A simple method for in situ freezing of anchorage-dependent cells including rat liver parenchymal cells; Ohno T et al.; We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish . The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at -80 degrees C . After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.

J Biol Chem, 1991 Feb 25, 266(6), 3688 - 94
Lymphocyte activation and phospholipid pathways . 31P magnetic resonance studies; Kaplan O et al.; 31P NMR spectra of perfused lymphocytes, embedded in alginate capsules and activated by interleukin-2, were remarkably different from those of control lymphocytes . The main differences were the appearance and gradual increase in phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine . These metabolic changes also occurred following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and were not dependent on a special growth medium . Nifedipine, a calcium channel blocking drug, inhibited the effects of phytohemagglutinin, but not of interleukin-2 . There were no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes . Thus, sustained accelerated turnover of phosphatidylcholine and phosphatidylethanolamine is an inherent feature of the activation process . 31P NMR spectra of lymphocytes are characterized by a low signal of phosphocholine . Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of cytidylyltransferase, indicated that choline kinase plays a key role in regulating phosphaditylcholine synthesis in human lymphocytes.

J Biol Chem, 1991 Feb 25, 266(6), 3586 - 93
Phosphatidate phosphatase from Saccharomyces cerevisiae . Isolation of 45- and 104-kDa forms of the enzyme that are differentially regulated by inositol; Morlock KR et al.; Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form of membrane-associated phosphatidate phosphatase from Saccharomyces cerevisiae (Lin, Y.-P., and Carman, G.M . (1989) J . Biol . Chem . 264, 8641-8645) revealed the existence of a 45-kDa form of the enzyme . Immunoblot analysis also showed that the 91-kDa form of the enzyme was a proteolytic product of a 104-kDa enzyme . The mitochondrial fraction contained the 45-kDa enzyme, whereas the microsomal fraction contained the 45- and 104-kDa enzymes . In vivo labeling experiments showed that the 104-kDa form of phosphatidate phosphatase was not a precursor of the 45-kDa form of the enzyme . The 45- and 104-kDa forms of phosphatidate phosphatase were purified and characterized . The enzymological properties of both enzymes were similar . However, the phosphatidate phosphatase 45- and 104-kDa proteins differed with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage . The expression of the phosphatidate phosphatase 45- and 104-kDa enzymes were regulated differentially in cells supplemented with inositol . The addition of inositol to the growth medium resulted in the induction of the phosphatidate phosphatase 45-kDa enzyme . The expression of the 104-kDa enzyme was not affected by inositol . Both forms of phosphatidate phosphatase were induced when cells entered the stationary phase of growth.

Eur J Biochem, 1991 Feb 14, 195(3), 653 - 61
Demonstration of a molybdenum- and vanadium-independent nitrogenase in a nifHDK-deletion mutant of Rhodobacter capsulatus; Schneider K et al.; In Rhodobacter capsulatus there exists, in addition to a conventional Mo-containing nitrogenase, a second, Mo-indendent nitrogenase which was demonstrated in wild-type cells as well as in cells of a nifHDK- mutant . To construct this R . capsulatus mutant, a 4-kb BglII-HindIII fragment encompassing nifK, nifD and most of the nifH coding region was substituted by an interposon coding for kanamycin resistance . The alternative nitrogenase is repressed by molybdenum . Mo concentration greater than 1 ppb in the growth medium prevented diazotrophic growth of nifHDK- cells and the expression of nitrogenase activity . The Mo-independent nitrogenase was maximally derepressed in activated carbon-treated media which contained less than 0.05 ppb Mo, high concentrations of iron (1 mM ferric citrate) and serine as N source . Under N2-fixing and optimal Mo-deficient conditions, nifHDK- cells grew with a doubling time of 9 h . The highest activity achieved with whole cells was 1.2 nmol ethylene.min-1.mg protein-1 . Vanadium neither stimulated nor inhibited growth and activity . The alternative nitrogenase reduced acetylene to both ethylene and ethane . With whole cells (nifHDK-) the proportion of ethane varied over 2-5% depending on the amount of residual traces of Mo in the medium . The addition of Mo to a growing, nitrogenase-active culture resulted in a slow decrease of total activity but also in a simultaneous increase of ethane production up to 40% . In contrast, cell-free extracts and the purified enzyme did not show any or only very little ethane formation (0-0.4%) . Both enzyme components appeared to be very labile proteins . Component 2 lost almost all its activity during cell breakage . With component 1 in crude extracts, if complemented with the stable component 2 of the Mo-nitrogenase from Xanthobacter autotrophicus, a recovery of 50% of the original whole cell activity could be achieved . During purification, component 1 (from the nifHDK- mutant) remained remarkably stable . The partially purified component 1 had a pH optimum (acetylene reduction) of 7.8-8.0, relatively high affinity to acetylene (Km = 0.055 mM) and was analyzed to contain 20 mol Fe atoms/mol protein, 0.2 mol Mo atoms and negligible amounts of V, W and Re . The dithionite-reduced dinitrogenase appeared to be ESR-silent . The results indicate that the alternative nitrogenase of R . capsulatus is not a vanadium enzyme but rather a heterometal-free Fe-nitrogenase or a nitrogenase with an as-yet-unidentified heterometal atom.

J Bacteriol, 1991 Feb, 173(3), 1331 - 4
Facultative alkaliphiles lack fatty acid desaturase activity and lose the ability to grow at near-neutral pH when supplemented with an unsaturated fatty acid; Dunkley EA Jr et al.; Two obligate alkaliphiles were found to have high levels of fatty acid desaturase, whereas two facultative alkaliphiles had no detectable activity . Supplementation of the growth medium of one facultative strain with palmitoleic acid, but not palmitic acid, at pH 7.5 inhibited growth . The obligate strain outgrows the facultative strain in a chemostat at a very high pH, whereas the converse is true at a pH of 7.5, and the two strains grow equally well at pH 9.0 . Thus, the obligate strain is compromised at a near-neutral pH but is better adapted than a related facultative alkaliphile to an extremely alkaline pH.

Biochim Biophys Acta, 1991 Feb 11, 1062(1), 13 - 8
Selection of hybrid plants obtained by electrofusion of vacuolated x evacuolated plant protoplasts in hypo-osmolar solution; Pfister E et al.; Vacuolated and evacuolated tobacco mesophyll protoplasts were electrically fused in hypo-osmolar media by using an alternating field of modulated amplitude for alignment . The vacuolated fusion partner was isolated from Nicotiana tabaccum L . cv Xanthi and the evacuolated one from the streptomycin-resistant strain Nicotiana tabaccum L . cv Petit Havana SR1 . The field and osmolarity conditions used ensured relatively high yields of heterologous fusion products despite the differences in density and size of the parental cells . After removal of the evacuolated, streptomycin-resistant fused and unfused protoplasts by flotation of vacuole-containing cells on iso-osmolar sucrose medium, the cybrids and hybrids were cultured in 25 microliters drops of agarose . During the first 5 weeks the non-fused Xanthi-protoplasts were used as a nurse culture . After addition of streptomycin to the growth media, cybrids and hybrids were successfully selected whereas fused and unfused vacuole-containing protoplasts died within 6 days . Only the streptomycin-resistant cybrids and hybrids developed into whole plants . On average a yield of 0.025% of streptomycin-resistant plants (referred to the total number of parental cells) was obtained . Polyacrylamide gel electrophoresis of leaf extracts of these plants showed that at least 50% of the streptomycin-resistant plants had a hybrid-esterase isoenzyme pattern . The protocol can be generalised by fusion of iodoacetamide-inactivated vacuolated protoplasts with meristematic (or evacuolized) protoplasts carrying no genetic marker . Use of evacolated protoplasts for electrofusion with vacuole-containing protoplasts therefore offers a way of overcoming the lack of suitable genetic markers for hybrid selection.

Biochim Biophys Acta, 1991 Feb 5, 1081(3), 279 - 84
Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast; Casey WM et al.; We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent . By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway . These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx . 50%, regardless of B-ring structure . We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect . A new procedure for the synthesis of ergosterol peroxides is also described.

Immunol Lett, 1991 Feb, 27(2), 119 - 25
The partially purified pre-implantation suppressor factor may be one of several factors to play a role in successful pregnancy; Bose R; Embryo-associated immunosuppressor factor (EASF) is detected by its suppressive properties in the concanavalin A (ConA)-induced lymphocyte proliferation assay . EASF was partially purified from human embryo growth media of in vitro fertilized ova (pre-implantation EASF) as three fractions . The aim of the present study was to determine whether the EASF isolated from human embryo growth media is similar to the EASF secreted by the pre-embryo, which has been shown to be associated with successful pregnancy . EASF activity was measured in the purified pre-implantation EASF fractions and in a total of 24 individual embryo growth media obtained from 10 patients undergoing in vitro fertilization and embryo transfer, where 6 patients achieved successful pregnancy and 4 did not . The results show that: (i) all three EASF fractions and the individual embryo growth media from patients who became pregnant were suppressive when added to the early phase of ConA-supplemented cultures; this was not seen with the embryo growth media from patients who failed to become pregnant, suggesting that the purified pre-implantation EASF may be one of several factors responsible for successful pregnancy; and (ii) some embryo growth media, irrespective of the pregnancy outcome of the patient, showed an irreversible immunosuppressive effect on ConA-induced lymphocyte proliferation, whereas none of the purified EASF fractions did; this could be due to the loss of activity during purification.

Int J Pept Protein Res, 1991 Feb, 37(2), 103 - 11
Peptides containing 2-aminopimelic acid . Synthesis and study of in vitro effects on bacterial cells; Le Roux P et al.; Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-Lys-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(Cl)-ambo-Apm and ambo-Apm-L-Ala(Cl) . In the two latter cases, Apm was associated with antibacterial amino acid beta-chloro-L-alanine {L-Ala(Cl)}, an inhibitor of alanine racemase and transaminase B . The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(Cl) had low MIC values in the presence of amino acids restoring protein synthesis . When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(Cl)-containing peptides, when the growth medium was supplemented with specific amino acids . It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.

J Gen Microbiol, 1991 Feb, 137 ( Pt 2), 315 - 21
Regulation of invertase in Aspergillus nidulans: effect of different carbon sources; Vainstein MH et al.; Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate . On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees . After this time the level of detectable invertase in the cultures declined . A proportion of the enzyme was secreted during the linear growth phase of the fungus . Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level . Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium . After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level . The repressive effect of other sugars, e.g . glucose and xylose, on invertase production was also demonstrated in this experimental system.

J Exp Zool, 1991 Feb, 257(2), 184 - 94
Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract; Hirobe T; Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin . Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating . On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes . From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts . The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested . In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture . FBS was added at a concentration of 1% for the first day . These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.

J Virol, 1991 Feb, 65(2), 641 - 8
Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells; Monroe SS et al.; Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis . We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification . Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium . This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells . During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection . This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa) . We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells . The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger . Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae.

Mol Chem Neuropathol, 1991 Feb, 14(1), 53 - 66
The metabolic fate of {3H-methyl}choline in cultured human neuroblastoma cell lines, LA-N-1 and LA-N-2; Singh IN et al.; The conversion of choline in cultures of the human neuroblastoma cell lines, LA-N-1 and LA-N-2 cells, was investigated in order to identify potential precursors in acetylcholine (AcCho) synthesis . LA-N-1, a catecholaminergic and LA-N-2, a cholinergic, cell line were incubated with {3H-methyl}choline (Cho) for varying periods of time up to 72 h . The radioactivity present in lipids and water-soluble metabolites increased linearly up to 24 h in both cell lines . Approximately 20% of the radioactivity associated with the water-soluble metabolites in both control (untreated) and retinoic acid-induced differentiated (RA-treated cells) LA-N-2 cells was present as Cho and AcCho . There was no detectable AcCho in the catecholaminergic cell line, LA-N-1 . The untreated and RA-treated LA-N-1 and LA-N-2 cells were labeled for 24 h with {3H-methyl}Cho, followed by a chase in growth medium containing 100 microM unlabeled choline . The distribution of radioactivity in the LA-N-2 cells was 6-10% of AcCho, 84-89% as phosphocholine (PCho), 1-3% as glycerophosphocholine (GroPCho), and 2-4% as Cho . The distribution of radioactivity in the LA-N-1 cells was similar except for the absence of AcCho . The distribution of radioactivity in the culture medium of LA-N-1 cells was 70-80% as Cho, 20-30% as PCho, and 1-3% as GroPCho . In contrast, the radioactivity was equally distributed between Cho (50%) and PCho (50%), with only 1-3% as GroPCho in the medium of LA-N-2 cells.

Cell Mol Neurobiol, 1991 Feb, 11(1), 143 - 56
Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme; Velan B et al.; 1 . Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter . The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase . 2 . The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51 . 3 . The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min . Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization . 4 . The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms . 5 . In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

J Bacteriol, 1991 Feb, 173(3), 1187 - 92
Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase; Styrvold OB et al.; It has been reported previously that Escherichia coli K-12 carries an amber mutation that prevents osmotic stress-dependent accumulation of trehalose (M . L . Rod, K . Y . Alam, P . R . Cunningham, and D . P . Clark, J . Bacteriol . 170:3601-3610, 1988) . We report that E . coli K-12 and W1485 (sup0) accumulated trehalose but that they required a higher osmotic strength in the growth medium than that required by their sup+ derivatives . Furthermore, the sup+ derivatives displayed both strongly increased trehalose-6-phosphate synthase activity and expression of otsA-lacZ and otsB-lacZ operon fusions compared with their parental strains . It is suggested that the amber mutation in question may be in a gene system encoding a transcriptional activator of the ots genes which govern the synthase . The much-used sup0 strain MC4100 behaved like the sup+ derivatives of W1485 with respect to trehalose synthesis . treA mutants with a defective periplasmic trehalase accumulated trehalose extracellularly under osmotic stress . The amount of trehalose excreted correlated with their synthase activity . Strains with an intact trehalase did not display extracellular trehalose accumulation . Thus, stressed E . coli cells regulate the cytoplasmic level of trehalose by a futile cycle involving overproduction, excretion, and degradation to glucose, which is reutilized.

Neurochem Res, 1991 Feb, 16(2), 137 - 44
Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures; Ferret B et al.; Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro . Under these conditions methylation processes measured with {3H} and {35S} methionine and {3H}ethanolamine as precursors showed an increased methylation of {3H}ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to {3H}choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine . A small increase in protein synthesis was observed after incubation of neurons with {3H}- and {35S}methionine . This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S-labeling in protein bands with moecular weights between 50 and 60 KDaltons . A protein band of about 55 KDaltons appeared to be preferentially labelled when {3H} methionine was the precursor . The treatment with gangliosides increased the incorporation of {methyl-3H} label after incubation of neurons with {3H} methionine, into total DNA and decreased that of total RNA . The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.

In Vitro Cell Dev Biol, 1991 Feb, 27A(2), 158 - 62
Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells; Goldstein B et al.; F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP) . We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA . In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents . When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA . However, when F9 cells were grown in Dulbecco's modified Eagle's medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells . 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM . These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium . Likewise, RA and cAMP both inhibited growth to the same extent in either medium . Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.

Appl Microbiol Biotechnol, 1991 Feb, 34(5), 559 - 64
Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture; Hayter PM et al.; Recombinant human interferon-gamma production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium . The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate . This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production . The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth . Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics . Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields . When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

J Biotechnol, 1991 Feb, 17(2), 169 - 76
Macroreticulate buffers: a novel approach to pH control in living systems; Righetti PG et al.; It is possible to control the pH of growing living systems in vitro by adding, to the growth media, macroreticulate buffers, i.e . amphoteric resins made with buffering and titrant groups simultaneously affixed to the matrix . Such beads possess a very precise isoelectric point (pI) and are able to maintain the solutions' pH close to their pI values for extended growth periods . These pearls are made of a neutral polyacrylamide backbone containing up to 200 mM grafted weak acrylamido acids and bases . It is possible to produce such buffers with any desired pH value in the pH 2.5-11 scale . An example is given of conditioning the pH of endive plants grown hydroponically.

Nucleic Acids Res, 1991 Jan 25, 19(2), 385 - 90
An automated method for DNA preparation from thousands of YAC clones; MacMurray AJ et al.; We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library . Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate . The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR) . Using this protocol, we produced 3000 YAC strain DNAs in three weeks . This automated procedure should be extremely useful in many genomic mapping projects.

FEMS Microbiol Lett, 1991 Jan 15, 61(2-3), 209 - 12
pH regulation of penicillin production in Aspergillus nidulans; Shah AJ et al.; As shown by both bioassay and high-performance liquid chromatographic (HPLC) analysis, penicillin G production by Aspergillus nidulans is subject to regulation by the pH of the growth medium . Penicillin titres were highest at alkaline pH and in strains carrying mutations in the regulatory gene pacC which mimics the effects of growth at alkaline pH . They were lowest at acid pH and in strains carrying mutations in the palA, palB, palC, palE or palF genes which mimic the effects of growth at acid pH.

J Biol Chem, 1991 Jan 15, 266(2), 1245 - 9
Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts; Jin P et al.; We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T . (1990) J . Cell Biol . 110, 1665-1672) . We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation . Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation . In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells . Inhibition of myoblast differentiation was, however, reversible . Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur . Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium . Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose . Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.

Biochim Biophys Acta, 1991 Jan 4, 1081(1), 61 - 4
Influence of aminophylline on the lipids in Microsporum gypseum; Bindra A et al.; The effect of aminophylline on the lipid synthesis of Microsporum gypseum has been examined . A decreased incorporation of {14C}acetate into lipids was observed when the cells were incubated for 1 h with aminophylline which was reflected in all the individual lipid fractions . However, cells grown with aminophylline in the growth medium exhibited increased levels of total phospholipids, which was probably due to a rise in intracellular cAMP as these cells exhibited 4-fold increased levels of cAMP . Decreased activity of phosphodiesterase by aminophylline accounts for the increased cAMP levels . Increased phospholipid content in aminophylline grown cells was further supported by the increased incorporation of {14C}acetate into phospholipids as well as increased activities of phospholipid biosynthetic enzymes in comparison to non-supplemented cells.

Gene, 1991 Jan 2, 97(1), 125 - 30
Induction of recombinant gene expression in Escherichia coli using an alkaline pH shift; Poindexter K et al.; A commonly used approach to control recombinant protein production in Escherichia coli utilizes the lambda pL promoter-operator and the lambda repressor . Inactivation of the lambda repressor function allows transcription to proceed . However, induction of the RecA-mediated cleavage of lambda repressor by the addition of nalidixic acid or inactivation of a temperature-sensitive lambda repressor by growth at the nonpermissive temperature can have detrimental effects on protein production . This paper describes the use of an alkaline shift in the pH of the growth medium that allows expression of genes from the pL promoter in a RecA-independent manner . This procedure results in high-level production of recombinant protein . The pH shift is performed in the stationary phase of cell growth, using culture volumes ranging from 1-1000 ml . This method can result in the production of over 15-fold more active protein than when using a temperature shift.

Nat Immun Cell Growth Regul, 1991, 10(1), 32 - 44
Effect of sex hormones on human T cell activation by concanavalin A; Yron I et al.; The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied . We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women . Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers . We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity . Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC . Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level . Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.

J Gen Microbiol, 1991 Jan, 137 ( Pt 1), 57 - 61
Helicobacter pylori catalase; Hazell SL et al.; Helicobacter pylori is the major aetiological agent of gastroduodenitis in humans . Due to the potential importance of catalase in the growth and survival of Helicobacter pylori on the surface of inflamed mucosae, we have characterized catalase from H . pylori as a prelude to further studies on the function of the enzyme in vivo . The catalase activity of H . pylori was significantly affected by the presence of blood, serum or erythrocytes in the growth medium: the greatest activity was expressed when the bacterium was grown on medium containing serum . H . pylori catalase is a tetramer with a subunit Mr of 50,000 . The enzyme had a pI of 9.0-9.3, was active over a broad pH range and was stable at 56 degrees C . It was non-competitively inhibited by sodium azide, and had no detectable peroxidase activity . The Km for the purified catalase was measured as 43 +/- 3 mM-H2O2 and the V as 60 +/- 3 mmol H2O2 min-1 (mg protein)-1 . The native catalase has absorption maxima at 280 nm and 405 nm with further minor shoulders or peaks at 510 nm, 535 nm and 625 nm, consistent with the presence of an iron-porphyrin prosthetic group.

J Gen Microbiol, 1991 Jan, 137 ( Pt 1), 213 - 7
Morphology of three polycentric rumen fungi and description of a procedure for the induction of zoosporogenesis and release of zoospores in cultures; Ho YW et al.; Three polycentric rumen fungi, LL, LC2 and Ruminomyces elegans (C2), isolated from the rumen of cattle were grown in six culture media . LL and LC2 were morphologically similar . Their characteristics resembled those of Orpinomyces and Neocallimastix joyonii, and they grew well and produced sporangia after 3-4 d growth in all the media . R . elegans differed morphologically from LL and LC2, but although it also grew well in all media, abundant sporangia occurred only after 2-3 d growth in media containing cellulose . Undifferentiated sporangia were produced by all three isolates; differentiation of the sporangia did not occur in the spent growth media . However, if thalli possessing recently-formed sporangia were transferred to, or flooded with, fresh liquid medium or rumen fluid, zoosporogenesis and liberation of zoospores occurred within 17-20 min for isolates LL and LC2 and 30 min for R . elegans . Procedures for inducing zoosporogenesis by polycentric anaerobic fungi are described.

Appl Environ Microbiol, 1991 Jan, 57(1), 85 - 92
Development of efficient suicide mechanisms for biological containment of bacteria; Knudsen SM et al.; To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids . In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number . We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions . The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization . The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment . As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function . With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function . Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically . These results permit the construction of extremely efficient biological containment systems.

Microbios, 1991, 65(264-265), 135 - 46
Comparative study of extracellular matrix protein binding to Aeromonas hydrophila isolated from diseased fish and human infection; Ascencio F et al.; Binding of 125I-labelled types I and IV collagen, fibronectin and laminin to Aeromonas cells is a common characteristic among Aeromonas strains isolated from diseased fish and human infections . The proportion of Aeromonas strains which bind to the various proteins were significantly greater for A . hydrophila than for A . sobria or A . caviae . The binding property was specific since it was inhibited by unlabelled homologous proteins . Bacterial cells incubated with proteolytic enzymes lose the ability of binding to 125I-labelled collagen type I and IV, fibronectin, laminin and vitronectin . A bacterial cell surface protein extract, containing active receptors competes with intact cells for 125I-extracellular matrix protein binding . Culture conditions greatly influence the expression of A . hydrophila cell surface binding structures for extracellular matrix proteins . The presence of calcium ions in the growth medium seems to be an important enhancer of the expression of extracellular matrix protein cell surface receptors.

Cytometry, 1991, 12(1), 64 - 7
Introduction of antibody into viable cells using electroporation; Berglund DL et al.; Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described . Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry . Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability . Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution . For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.

Int J Radiat Oncol Biol Phys, 1991 Jan, 20(1), 59 - 64
Effect of external pH on heat sensitization by local anesthetics; Coss RA et al.; The sensitization of asynchronous Chinese hamster ovary (CHO) cells to 43 degrees C by procaine, lidocaine, and tetracaine was examined, with the pH of the medium carefully controlled at approximately pH7 and 8 . Thermal enhancement factors for 43 degrees C were calculated for the surviving fraction of 0.01 . The thermal enhancement factors of the local anesthetics were increased at approximately pH8 by factors of 2-12, depending on the local anesthetic and its concentration . The concentration of the uncharged free base form of the local anesthetic in the culture medium correlated positively with the thermal enhancement factors of each local anesthetic and in a near linear fashion with the thermal enhancement factors for procaine . However, the concentration of the cationic form of the local anesthetics in the growth medium did not correlate with heat sensitization . We conclude that the ability of local anesthetics to sensitize cells to heat killing is dramatically influenced by the extracellular pH, with increased sensitization at the more basic pH's . Secondly, it is the extracellular concentration of the free base form of the local anesthetics that correlates with heat sensitization.

Cancer Res, 1991 Jan 1, 51(1), 37 - 42
Concentration-dependent increase of murine P388 and B16 population doubling time by the acyclic monoterpene geraniol; Shoff SM et al.; Geraniol, an acyclic end product of a plant isoprene pathway and a pyrophosphorylated intermediate in plant and animal pathways, caused a concentration-dependent increase in the population doubling time of murine P388 leukemia cells in suspension culture and of B16 melanoma cells in monolayer culture . The suppression of the growth of P388 cells by geraniol (0-0.9 mM) and by mevinolin (0-0.25 microM), a competitive inhibitor of mevalonate biosynthesis, was reversed by the addition of 0.5 mM mevalonolactone to the growth medium . Flow cytometry of asynchronous B16 cells grown with geraniol (0-0.15 mM) revealed a population characterized by larger cells with altered nuclear characteristics . Over the course of four studies, dietary geraniol increased the 50% survival time of mice by 10, 29, 33, and 50% following the i.p . transfer of P388 cells . The results of the latter study showed that, following the i.p . transfer of 1 x 10(5) P388 cells, the control group of female C57BL x DBA/2 F1 mice had a 50% survival time of 24 days and a maximum survival of 27 days . Mice fed a diet containing 0.1% geraniol for 14 days prior to and following the P388 cell transfer had a 50% survival time of 36 days, and 20% of the mice remained free of tumors during the 50-day trial . These studies support the possibility that monoterpenes and other isoprenoid products of plant metabolism are in part responsible for the anticarcinogenic actions of diverse fruits, vegetables, and cereal products.

Int J Dev Neurosci, 1991, 9(4), 339 - 45
Elevated potassium prevents neuronal death but inhibits network formation in neocortical cultures; Baker RE et al.; Chronic depolarization is inimical to neuronal growth and synaptogenesis so that spontaneous action potential generation appears to be required for the normal cytomorphological maturation of neocortical networks . The efficacy of 25 mM K in suppressing spontaneous bioelectric activity was monitored by extra- and intracellular recording from the explants . Intracellular recording from individual neurons showed that membrane potentials were reduced to ca -30 mV in potassium cultures but rapidly repolarized to ca -50 mV when returned to normal growth medium . Though action potentials could be readily evoked from these explants, spontaneous discharges and postsynaptic potentials were absent from potassium-treated cultures . Both spontaneous bioelectric activity and postsynaptic potentials returned to the cultures by 5 days after returning the explants to normal growth medium . Extracellular recordings also showed that the explants were bioelectrically silent in the presence of 25 mM K or 25 mM K plus tetrodotoxin . In contrast to tetrodotoxin alone, bioelectric activity was absent when the cultures (with or without tetrodotoxin) were returned to normal growth medium . The explants gradually began to evince spontaneous bioelectric activity between 3 and 5 days after being returned to normal growth medium . Massive cell death induced by chronic exposure to tetrodotoxin was totally prevented by concomitant addition of 25 mM potassium, though these explants were significantly thinner than controls due to a large decrease in neuropil . We conclude that chronic depolarization of neonatal cortical explants by potassium results in a delayed return of spontaneous bioelectric discharges . Chronic depolarization results in a retardation of network formation in these explants apparently due to a lack of neurite and/or synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Dev Neurosci, 1991, 9(4), 321 - 9
Chronic blockade of bioelectric activity in neonatal rat neocortex in vitro: physiological effects; Baker RE et al.; We have examined what effect the loss of spontaneous bioelectric activity has on neural network formation in organotypic rat neocortical explants grown under serum-free culture conditions . Explants were taken from dorsal midline (presumptive visual) and lateral (presumptive auditory) occipital cortex and chronically exposed to tetrodotoxin which blocked all measurable bioelectric activity between change of medium . Extracellular recordings revealed complex, rhythmic spontaneous and evoked multiunit discharges in all explants examined (following tetrodotoxin washout in the experimental group) . Control auditory explants had significantly more sites from which electric activity could be recorded compared with control visual explants . Auditory cultures showed no effect of the tetrodotoxin treatment, whereas visual explants showed significant increases over control values, equalling the auditory values . This increased level of spontaneous bioelectric activity was maintained for at least 10 days following transfer of the cultures to control growth medium . There was no significant difference between control visual and auditory explants regarding the number of sites from which evoked activity was seen . Nor did either cortex group show an effect of tetrodotoxin on the number of sites from which evoked activity was seen . The frequency with which spontaneous bioelectric discharges occurred per site increased with age in auditory vs visual cortex . These differences, however, were abolished in the tetrodotoxin-treated groups . It was concluded that neocortical explants which have experienced chronic suppression of spontaneous electric activity did not suffer deficits in neural network formation, though there is an effect on the incidence and frequency with which such activity is given.

Environ Mol Mutagen, 1991, 18(1), 22 - 7
Cytocidal and filamentous response of Escherichia coli cells exposed to low concentrations of hydrogen peroxide and hydroxyl radical scavengers; Brandi G et al.; The hydroxyl radical scavengers thiourea, ethanol, and dimethyl sulfoxide significantly reduced the killing of Escherichia coli elicited by low concentrations of H2O2 (resulting in mode 1 killing) when treatments with the oxidant and the scavengers were performed in a complete growth medium (K medium) but not in M9 salts . In addition, thiourea efficiently prevented the toxic response to high concentrations of H2O2 (resulting in mode 2 killing) under both exposure conditions . Sod A cells, which do not respond with a bimodal pattern of toxicity when challenged with increasing concentrations of H2O2, were markedly protected by thiourea against the lethal action of various levels of the oxidant (which in the wild-type strain result in either mode 1 or mode 2 killing) under conditions of treatments in both K medium and M9 salts . In some experiments, wild-type cells were challenged with a low concentration of H2O2 (in the absence or presence of hydroxyl radical scavengers) and then postincubated in fresh K medium for various time intervals . It was found that scavengers were able to inhibit the filamentous response generated by exposure to the oxidant in K medium . Both the length and the number of filaments were markedly reduced . Treatment in M9 salts resulted in a limited number of very short filaments, and this response was slightly reduced by the scavengers.

Artery, 1991, 18(3), 115 - 24
Antioxidants stimulate endothelial cell proliferation in culture; Kuzuya M et al.; The effect of antioxidants on the proliferation of cultured endothelial cells (ECs) were studied . Probucol, a lipid-lowering drug with antioxidant properties, added to the growth medium stimulated the proliferation of ECs . D1-alpha-tocopherol (tocopherol), a natural antioxidant, and butylated hydroxytoluene, a synthetic antioxidant also had the same effect on the proliferation of ECs . There was no significant difference in the content of thiobarbituric acid-reacting substances between the probucol-containing growth medium and control medium during the growth assay . Pretreatment of ECs with probucol or tocopherol also enhanced ECs proliferation in medium without any added antioxidants . The addition of free radical scavengers superoxide dismutase, catalase and mannitol to the growth medium did not enhance the proliferation of ECs . These findings indicate that the stimulatory effect on ECs proliferation of antioxidants in the growth medium appears to be unrelated to preventing free radical reactions in the medium itself, but rather may be related to the uptake of antioxidants by ECs.

Life Sci, 1991, 49(16), 1173 - 81
Infection of cultured human airway epithelial cells by influenza A virus; Reiss TF et al.; The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway . We report in vitro infection of human airway epithelial cells by influenza A virus . Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection . All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus . No hemadsorption was detected with the WSN virus . One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN . Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells . Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins . Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant . Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time . The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.

Symp Soc Exp Biol, 1991, 45, 21 - 9
The phenotype of Arabidopsis thaliana det1 mutants suggests a role for cytokinins in greening; Chory J et al.; When grown in the absence of light, the det1 mutants of Arabidopsis thaliana (L.) Heynh . develop characteristics of light-grown plants as determined by morphological, cellular, and molecular criteria . Further, in light-grown plants, mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and the chloroplast developmental program . Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology . Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally 'light'-regulated . These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate abnormal cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants.

J Clin Periodontol, 1991 Jan, 18(1), 26 - 9
Fluctuation of the microbiota of the tongue in humans; van der Weijden GA et al.; The purpose of this study was to examine whether variations occur in the % of motile micro-organisms on the dorsum of the tongue during a 1-month time-period and further to examine 2 transport fluids: the growth medium BM and reduced transport fluid (RTF), for their ability to conserve motility of microorganisms in comparison to sterile saline . Both transport fluids showed a significant reduction in the % of motile micro-organisms within 15 min . The use of RTF resulted, during a 60-min period, in a significantly higher % of motile micro-organisms compared to sterile saline . Results showed that the % of motile micro-organisms on the dorsum of the tongue was not constant . A significant reduction in the % of motile micro-organisms was observed in the course of the month involved in this study.

J Physiol (Paris), 1991, 85(3), 154 - 7
Cholinergic fiber growth in co-cultures of CNS tissue; Gahwiler BH et al.; In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells . To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity . Under these conditions, considerably fewer cholinergic cells appeared to survive . A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures . Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures . These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue . To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum . In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue . This failure might in part be related to the lack of trophic factors released by the target tissue . We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF . Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1991, 157(1), 49 - 53
Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids; Keweloh H et al.; In the presence of sublethal concentrations of phenol, 4-chlorophenol, and p-cresol in the growth medium, cells of Escherichia coli modified the fatty acid composition of their lipids . The results of these changes was an increase in the degree of saturation of lipids probably in order to compensate an increase of fluidity of the membrane induced by the phenols . Supplementation of the growth medium with saturated fatty acids could also enhance the degree of lipid saturation due to the incorporation of the acyl chains in the phospholipids . At the same time the growth of cells was less inhibited than in unsupplemented cells . The increase of tolerance of cells by manipulating the lipid composition indicates that the membrane structure plays a crucial role in the mode of action of phenols.

Arch Microbiol, 1991, 156(1), 38 - 42
Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress; Singh KK et al.; When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (aw 0.997) to a medium containing 8% NaCl low water activity growth medium (aw 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude . Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced . Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S . cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited . Potassium ions accumulated in S . cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period . The trehalose content also increased in adapting cells . It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibition of glycolysis, glycerol being produced glycolytically in S . cerevisiae . The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S . cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.

Genetica, 1991, 84(3), 145 - 54
Micro-spatial population differentiation in activity of glycerol-3-phosphate oxidase (GPO) from mitochondria of Drosophila melanogaster; Ross JL et al.; Replicate mass-bred laboratory populations of D . melanogaster were derived from females collected in the Tahbilk winery cellar and from females collected outside but from within two kilometers of the cellar . When mitochondrial extracts from larvae were assayed for specific activity of glycerol-3-phosphate oxidase the cellar populations had levels only 50% of those from the outside area, confirming an earlier report of such a difference among isofemale lines derived from these same areas . This micro-spatial differentiation occurred when larvae were raised on a medium supplemented with both sucrose (5% w/v) and ethanol (4% v/v), known to effect high GPO activity, but was not detected when the larvae were raised on unsupplemented medium . A heritable basis for larval GPO activity variation was confirmed in a set of 32 isogenic second chromosome substitution lines and measured in a subset of 4 of these lines about 25 generations later . A reciprocal cross using two isogenic substitution lines with the highest and lowest activities suggested the difference was attributable to genes acting additively and that there were no maternal or paternal effects . The detection of a collection site difference in GPO enzyme activity in the isogenic lines suggests that polymorphic variation on the second chromosome is responsible for the differentiation at the winery . Variation in adult GPO activity did not show a dependence on the winery location from where the isogenic lines were derived nor was there an effect of line . Adult GPO activity was significantly higher than that detected in larval tissues and did not show a dependence on the sugar/ethanol level in the growth medium.

Oncology, 1991, 48(6), 469 - 73
Modulation of methotrexate cytotoxicity with natural interferon upon human leukemia cell line HL-60; Sur P et al.; The effect of alpha- and gamma-interferon on methotrexate cytotoxicity against human promyelocytic cell line HL-60 has been evaluated . Synergistic inhibition of proliferation is observed with the combination of methotrexate and gamma-interferon . Enhanced cytotoxic effect of methotrexate with alpha- or gamma-interferon is removed by adding thymidine to the growth medium . The depletion of folate pools caused by methotrexate is enhanced in presence of interferons, this depletion is also removed by adding thymidine to the growth media . Synchronization of HL-60 cells in 'S' phase of cell cycle caused by methotrexate is enhanced in the presence of interferons . These results suggest that specially gamma-interferon changes the cellular environment of HL-60 cells in such a way as to make methotrexate more potent cytotoxic agent to HL-60 cells causing cell death . This study also clearly indicates the biochemical role of thymidine in modulating the activity of methotrexate in combination with interferons.

Tissue Cell, 1991, 23(6), 763 - 75
Antiport mediated rounding and endocytosis are enhanced by sulphate; Sit KH et al.; Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive . At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity . This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width . Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion . Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages . However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz . epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202) . The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change . However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME . FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions . Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches . AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting . It appears to be not a simple endocytosis-exocytosis pathway.

Lab Delo, 1991, (12), 47 - 9
{The effect of sodium nucleinate on the proliferative and specific activity of hybridoma cells in vitro}; Fedorova VA et al.; Sodium nucleinate, an officinal preparation (Na PHK), added to the growth medium in concentrations of 10 to 50 micrograms/ml enhanced the growth of hybridoma cells in vitro . Na PHK presence in the cultivation medium in the above concentrations enhanced the specific antibody-producing activity of hybridomas.

Int Arch Allergy Appl Immunol, 1991, 95(2-3), 248 - 56
Faecally derived hydrolytic enzymes from Dermatophagoides pteronyssinus: physicochemical characterisation of potential allergens; Stewart GA et al.; The previous findings that the group I and III mite allergens, and amylase present in mite faeces are hydrolytic enzymes has prompted a study to determine whether this material contains other enzymes which could be allergenic . Thus, spent growth medium devoid of whole Dermatophagoides pteronyssinus mites was shown to contain glucoamylase, lipase and lysozyme in addition to the cysteine protease, serine protease and amylase activities associated with the above allergens, respectively . All of these enzymes are probably associated with mite digestive processes . They were rapidly solubilised, heterogeneous with regard to charge (pI in the range 4-8) and demonstrated maximum biochemical activity in the neutral pH range . Three serine proteases were detected and comprised a chymotrypsin-like, a trypsin-like and an unclassified enzyme with pI of 4.1 and 5.3, 8.5 and 7.1, respectively . Only one cysteine protease was observed, which paralleled immunochemically identified Der p I in a variety of assays . It was shown to cleave at lysyl residues and could be inhibited by the specific cysteine protease inhibitor, E-64 . The remaining serine proteases, glucoamylase, lipase and lysozyme represent potential allergens.

Int Arch Allergy Appl Immunol, 1991, 94(1-4), 357 - 8
Allergenicity and physicochemical characterization of house dust mite derived amylase; Lake FR et al.; The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus . In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I . Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000 . The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME . It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic . The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds.

J Med Vet Mycol, 1991, 29(3), 165 - 77
Variable colonial phenotypic expression and comparison to nuclei number in Blastomyces dermatitidis; Clemons KV et al.; We examined colonial phenotypes of five isolates of Blastomyces dermatitidis at 33, 35 and 37 degrees C on four growth media . Three different colony types were identified: yeast, mycelial, and a mixed type consisting of both yeast and mycelia . Each isolate varied in its ability to grow on the different media and at different temperatures, and in the types of colonies it produced in the various temperature-media combinations . Quantification of the number of nuclei per yeast cell by fluorescent staining revealed no correlation between the number of nuclei per cell and the colonial phenotype . These results indicate that the colonial phenotype of B . dermatitidis varies with the isolate as well as with temperature and culture medium, but is not correlated with the number of nuclei per yeast . These findings could provide a start towards typing B . dermatitidis isolates.

Mikrobiologiia, 1991 Jan-Feb, 60(1), 65 - 70
{Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate}; Kaliuzhnaia VM et al.; The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e . under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP) . The stimulating action of exogenous cAMP was found only in a medium with salts and glucose . The induction by IPGP was highest during the growth in a medium with glucose and maltose . When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth . The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E . coli: the expression of catabolite-sensitive E . coli genes was subject to both positive and negative regulation in one and the same inducible system . The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.

Neirofiziologiia, 1991, 23(2), 199 - 205
{Action of omega-conotoxin on calcium currents of the rat pituitary GH3 cell line}; Fomina AF et al.; Whole-cell modification of the patch clamp method was used to examine the action of omega-CgTX on calcium currents in GH3 pituitary cells . Two quite distinct components of inward calcium currents were observed in the presence of 15 mmol/l of calcium in the external solution . One was activated from the holding potential -80 mV by testing pulses more positive than -50 mV . The shift of the holding potential to -40 mV resulted in the stationary inactivation of this low voltage activated current component . It was found that omega-CgTX activated both low-threshold and high-threshold calcium currents at the first moment of application, but low-threshold current component increased more significantly . Full effect was developed for less than 30 s . Then time decay of currents was comparable with that of the "wash-out" process . Incubation of cells in the growth medium that contained 5 mumol/l omega-CgTX during 2 hour induced an increase in density of both types of calcium currents, then it fell after 2 hours of incubation in the same medium.

Histochemistry, 1991, 95(5), 427 - 33
Differences in tetranectin immunoreactivity between benign and malignant breast tissue; Christensen L et al.; Tetranectin (TN) is a human, plasminogen kringle 4 binding plasma protein with ubiquitous cellular distribution and lectin-like characteristics . By means of the peroxidase-antiperoxidase staining technique a polyclonal and a monoclonal antibody were used to demonstrate TN within the intracellular as well as the extracellular compartment of invasive breast carcinoma . Whereas cell associated TN was universal showing only quantitative differences depending of the growth pattern of the tumor, 78 of 133 tumors displayed TN extracellularly as well . The occurrence of this stromal TN immunoreactivity was closely associated with desmoplasia, recognized morphologically by an increase in fibroblastic cells and immunohistochemically by an intense staining for the connective tissue glycoprotein fibronectin (FN) . Benign breast tissue displayed a universal, intense cytoplasmic but no extracellular reaction for TN, with the exception of rare foci of granulation tissue and around dilated cysts . Functional studies have shown that human embryonal lung fibroblasts increase their release of TN to the growth medium upon stimulation . The presence of TN extracellularly within fibroblast-rich foci of desmoplasia (and granulation tissue) suggests that a similar increased release of the protein takes place in vivo during active states . Desmoplasia has been found to have a protective effect on tumor cell propagation and metastasis in a murine model . The molecular interactions, which are responsible for this effect, are undoubtedly complex . However, TN may, by its specific binding to kringle 4 of plasminogen and its high affinity for sulphated polysaccharides, add to the understanding of how plasminogen activation is modulated at the local extracellular level.

J Biol Chem, 1990 Dec 25, 265(36), 22210 - 6
Expression of sodium-linked nucleoside transport activity in monolayer cultures of IEC-6 intestinal epithelial cells; Jakobs ES et al.; Mature, confluent monolayer cultures of IEC-6 rat intestinal epithelial cells in conventional growth media express both Na(+)-linked, concentrative nucleoside transport (NT) activity and equilibrative, inhibitor-sensitive NT activity, but do not show morphologic differentiation . Na(+)-dependent fluxes of Ado and formycin B were minor in early subconfluent IEC-6 monolayers, but increased severalfold to become the major component of influx of these agents in confluent monolayers grown in medium containing Nu-Serum, a commercial medium supplement with a low serum content . In monolayers cultured in medium with fetal bovine serum, cell proliferation rates were similar to those in medium supplemented with Nu-Serum, but expression of Na(+)-linked NT activity was 6-8-fold lower than in monolayers grown in the latter medium . Inclusion of hydrocortisone in growth medium with Nu-Serum caused a 2-fold increase in the expression of Na(+)-linked NT activity . Experiments in which components of medium supplementation were withheld showed that insulin and epidermal growth factor were important in expression of the Na(+)-linked NT activity . Because the Na(+)-linked NT system has a brush border location in fresh intestinal epithelium, it is concluded that the regulated expression of this activity in the IEC-6 monolayers is a differentiative change.

J Steroid Biochem Mol Biol, 1990 Dec 20, 37(6), 1097 - 101
Manipulation of cell cycle kinetics: influence on the cytotoxicity of doxorubicin in human breast cancer cells; Bontenbal M et al.; In vitro exposure of estrogen receptor-negative (ER-) EVSA-T human breast cancer cells to insulin and/or estradiol had no effect on cell cycle distribution, in contrast to a 3-5-fold increase in the percentages of cells in the S-phase of the cell cycle in the ER+ MCF-7 cell line . Estrogen pretreatment of MCF-7 cells followed by incubation with doxorubicin resulted in an augmented inhibition of cell growth compared to unstimulated controls . This delay in growth was accompanied by a decrease in the percentages of cells actively synthesizing DNA, and by an augmented percentage of cells exhibiting a G2M-amount of DNA at the end of a 6-9 day period of culture in complete growth medium.

Biochem Biophys Res Commun, 1990 Dec 14, 173(2), 741 - 7
Expression and secretion of goat alpha-lactalbumin as an active protein in Saccharomyces cerevisiae; Takeda S et al.; An expression plasmid for goat alpha-lactalbumin in Saccharomyces cerevisiae, pSKA100, was constructed into a shuttle vector, pYG100, by inserting cDNA which encodes goat pre-alpha-lactalbumin and two-thirds of the 3'-non-coding region . The goat alpha-lactalbumin was expressed under the yeast glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and terminator of pYG100 and secreted in the growth medium for yeast as a precise mature protein, possessing specific activity essentially the same as that of authentic goat alpha-lactalbumin in lactose synthesis.

Biochim Biophys Acta, 1990 Dec 4, 1047(3), 290 - 3
Effect of the methylation inhibitors 3-deazaadenosine and 3-deazaaristeromycin on phosphatidylcholine formation in Tetrahymena; Smith JD et al.; The methylation inhibitors 3-deazaadenosine and 3-deazaaristeromycin inhibited the methylation pathway for phosphatidylcholine formation in Tetrahymena . At the same time, the phosphatidylcholine levels within the cell were maintained by increased use of the phosphotransferase pathway . Cell growth was not affected at inhibitor concentrations up to 50 microM but was 50% inhibited at inhibitor concentrations of 100 microM . This growth inhibition was not reversed by the addition of choline to the growth medium . The added choline resulted in greater use of the phosphotransferase pathway, even in uninhibited cultures, but there was no effect on phosphatidylcholine levels, pointing to a much tighter control on phosphatidylcholine formation than exists in animal cells.

Br J Haematol, 1990 Dec, 76(4), 476 - 83
Interleukin-6 is a cofactor for the growth of myeloid cells from human bone marrow aspirates but does not affect the clonogenicity of myeloma cells in vitro; Borinaga AM et al.; Several groups have claimed that IL-6 is a growth factor for human myeloma cells in vitro . Bone marrow aspirates from 30 patients at different stages of treatment with VAMP/high dose melphalan, were examined for myeloma colony formation (MY-CFUc) using a clonogenic assay in vitro . Myeloma cells from 16/30 patients produced MY-CFUc in our assay system, which uses heavily irradiated HL60 cells as an underlay in soft agar . These heavily irradiated cells were shown to be essential for the inhibition of granulocyte-macrophage colonies (GM-CFUc) . The addition of recombinant human IL-6 (10 ng/plate) reduced the number of bone marrow samples which produced MY-CFUc from 16 to six . Furthermore, the addition of antibody to IL-6 (1 microgram/plate) failed to inhibit MY-CFUc from 6/7 samples . Conditioned medium from human peripheral blood mononuclear cells (PBMC-CM) contains approximately 2 ng/ml IL-6 and can be used to stimulate the growth and maintenance of the B9 murine IL-6 dependent hybridoma cell line . Recombinant human IL-6 supported the growth of B9 cells in a clonogenic assay and growth was inhibited by anti-IL-6 in the presence of rhIL-6 or PBMC-CM . Mononuclear cells from a second group of myeloma patients were cultured in soft agar in a mixture of PBMC-CM and fresh growth medium . Nine of the 10 samples produced myeloid colonies which consisted of granulocytes, monocytes and macrophages and the number of colonies was reduced by at least 50% in 6/8 samples when anti-IL-6 was added to the cultures . In no instance were MY-CFUc produced . Also, conditioned medium from the bladder carcinoma cell line 5637, which is used routinely as a source of granulocyte-macrophage colony stimulating factor (GM-CSF), contains approximately 4 ng/ml IL-6 . Although rhIL-6 failed to stimulate GM-CFUc in the absence of other growth factors, addition of anti-IL-6 to cultures containing a suboptimal amount of 5637-CM reduced the number of colonies by 50% . These data provide evidence that IL-6 is a cofactor for the growth of myeloid precursors but does not affect the proliferation of human myeloma cells in vitro.

Biochimie, 1990 Dec, 72(12), 893 - 5
Regulation of a thermostable alpha-amylase of Streptomyces thermoviolaceus CUB74: maltotriose is the smallest inducer; Bahri SM et al.; We have examined induction and repression by various sugars and carbon sources of the synthesis of a thermostable alpha-amylase in its natural host, S thermoviolaceus CUB74 . The smallest molecule capable of inducing synthesis of the enzyme was maltotriose whereas maltose had no effect which might suggest a different control system from that found in other streptomycete amylases . Addition of mannitol to the growth medium impeded the alpha-amylase induction whereas glucose had no effect . After cloning of its gene into a new streptomycete host, S lividans TK24, the S thermoviolaceus alpha-amylase could not be induced by any of the sugars tested.

Mol Biochem Parasitol, 1990 Dec, 43(2), 221 - 30
Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates; Beck JT et al.; The nutritional requirements for folates and pterins were assessed for two strains of Leishmania donovani promastigotes, a wild-type (D1700) parental strain and a mutant derivative (MTXA5) which possesses a markedly diminished capacity to transport both {3H}folate and {3H}methotrexate (MTX) . Both L . donovani strains have an absolute growth requirement for an exogenous pterin, since their proliferation could not be sustained in completely defined medium lacking either a pterin or a folate . Supplementation of the growth medium by many of a spectrum of folates and pterins could support growth of the wild-type cell line . Surprisingly, however, the MTXA5 strain could not thrive in folate-deficient medium fortified with any of the pterins that promoted wild-type cell division, including biopterin and neopterin . The relationship between the incapacity of MTXA5 cells to transport folate and methotrexate and their inability to multiply in folate-deficient medium supplemented with pterins was evaluated using genetic approaches . First, an independently generated MTX-resistant mutant, MTXB4, was isolated in 1 mM MTX . The phenotype of the MTXB4 cells was like that of MTXA5 cells since they were unable to transport {3H}MTX and {3H}folate or grow in folate-deficient medium supplemented with pterins . Second, three revertants of the MTXA5 cells were selected for their ability to grow in 1 microM biopterin . All three revertants had regained {3H}folate and {3H}MTX transport capability concomitant with growth sensitivity to methotrexate toxicity . These observations provide genetic evidence that the competence of L . donovani to transport {3H}folate and {3H}MTX and the capability of the organisms to utilize pterins as a nutritional factor are influenced by a common genetic locus.

J Gen Microbiol, 1990 Dec, 136 ( Pt 12), 2471 - 80
Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153); Brownlie L et al.; The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied . In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-1 than at 0.15 h-1 . In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host . Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene . Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium . The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage . We conclude that in complex media the plasmid is maintained longer by E . coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used . A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.

Mol Immunol, 1990 Dec, 27(12), 1319 - 24
Receptor-like oncogenes: functional analysis through novel experimental approaches; Yarden Y; A growing proportion of the known protooncogenes encode putative receptors for growth/differentiation factors . The detection and identification of the hypothetical ligands of these presumed receptors, and the elucidation of their biological roles constitute a new biochemical challenge . Employing the neu protooncogene, here three experimental approaches to these problems are described . Stimulatory anti-receptor antibodies appear to mimic the action of the presumptive ligand of the neu receptor, and also lead to the conclusion that the oncogenic receptor, unlike the normal p185neu, is functionally equivalent to a ligand-stimulated receptor . Second, an experimental strategy was developed for the detection of the neu ligand . Employing this approach a candidate ligand was detected in the growth medium of certain oncogene-transformed fibroblasts . Thirdly, engineered chimeras of the neu gene and the gene for the EGF-receptor enabled construction of a neu receptor that is responsive to a heterologous ligand . Combinely, these approaches may provide a detailed biological picture of the action of the putative ligand, and may be generally applicable in the study of other receptor-like oncogenic proteins.

Zentralbl Bakteriol, 1990 Dec, 274(3), 426 - 32
Inducible expression of herpes simplex virus type 1 glycoprotein C in NIH 3T3 cells; Kleim JP et al.; Antibodies directed against the glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) are known to be mainly HSV-1-specific, whereas antibodies against other major HSV-1 glycoproteins cross-react with HSV-2 antigens . To investigate the immunological features of gC-1, the gene encoding gC-1 was isolated and cloned from DNA of cells infected with HSV-1 . The 3.6 kbp SalI fragment R of HSV-1 DNA was modified in order to place the gene under transcriptional control of the glucocorticoid dependent promoter of the MMTV-LTR . NIH 3T3 cells were transfected with the resulting plasmid . Cell lines established by selection for the vector-encoded marker gene were tested for the ability to synthesize gC-1 after addition of dexamethasone to the growth medium . Glycoprotein-enriched cell extracts of several clones were shown to contain gC-1 by immunoblotting using a gC-1-specific monoclonal antibody . One cell line was used to show the presence of gC-1 also in the culture supernatant . gC-1 synthesis decreased after several passages of the cells but could be restimulated by the addition of 5-azacytidine to the cultures.

J Cell Physiol, 1990 Dec, 145(3), 409 - 13
Significance of extracellular zinc-binding ligands in the uptake of zinc by human fibroblasts; Ackland ML et al.; Extracellular zinc (Zn)-binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts . The uptake of alpha 2-macroglobulin, a major serum Zn-binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate . The fibroblast growth medium, BME with 10% FBS, contains several Zn-binding ligands . These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator . Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW less than 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration . Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration . When histidine and desferrioxamine, two structurally different Zn-binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (+/- S.E.M.) of 373 +/- 6 pmol/micrograms DNA/h and 0.08 +/- 0.004 microM for histidine, and 349 +/- 10 pmol/micrograms DNA/h and 0.06 +/- 0.008 microM for DFO, suggesting that the same transport mechanism was operating in both systems . We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that "free" Zn is acquired by the cell.

Cancer Res, 1990 Dec 1, 50(23), 7650 - 61
Effect of epidermal growth factor on the cellular proliferation and phenotype of a neoplastic human salivary intercalated duct cell line or its derivatives; Aladib W et al.; A neoplastic human salivary intercalated duct cell line (HSG) and its derivatives, HSG with a myoepithelial cell phenotype (HSG-AZA1) and HSG with an acinar cell phenotype (HSG-AZA3), which were induced by 5-azacytidine treatment of HSG cells, were cultivated in the presence of epidermal growth factor (EGF) . Morphological changes occurred; cells that were spindle shaped or stellate and had long cytoplasmic processes appeared in all of the treated cells . Major alterations, such as expression of neuron-specific enolase, neurofilaments, or synaptophysin as well as formation of neurite-like structures densely packed with microfibrils and microtubules, were observed in these cells with a phenotype similar to that of neuron-like cells . Both the anchorage-independent and anchorage-dependent growths of the treated HSG cells and only anchorage-dependent growth of the treated HSG-AZA1 cells were suppressed, whereas the anchorage-dependent growth of the treated HSG-AZA3 cells was enhanced according to the increasing concentrations of EGF . In addition, it has been found by statistical analysis that the growth of HSG cells in culture is suppressed and that of HSG-AZA3 cells in culture is stimulated according to the increasing concentrations of EGF added, whereas the growth of cultured HSG-AZA1 cells is hardly affected by EGF treatment . The HSG, HSG-AZA1, and HSG-AZA3 cell had 1.8 x 10(5), 1.83 x 10(5), and 1.34 x 10(5) EGF receptors, respectively . The amounts of the EGF internalized into cells were larger in HSG-AZA3 cells than in HSG or HSG-AZA1 cells . These findings indicate that the conversion of HSG, HSG-AZA1, and HSG-AZA3 cells into neuron-like cells occurs in growth medium containing EGF, with a concomitant modulation of the growth potentials of the cells which were examined in a different manner.

Agric Biol Chem, 1990 Dec, 54(12), 3111 - 6
Expression and secretion of bovine beta-lactoglobulin in Saccharomyces cerevisiae; Totsuka M et al.; A bovine beta-lactoglobulin (beta-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-beta-LG cDNA and secreted into its growth medium . The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-beta-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector . In the supernatant of the yeast growth medium, beta-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l . A Western-immunoblotting analysis revealed that beta-LG secrected in the growth medium had the same mobility as that of authentic bovine beta-LG . The N-terminal sequence was also identical with that of authentic mature bovine beta-LG.

J Biol Chem, 1990 Nov 5, 265(31), 19047 - 52
The gene coding for the yeast oligomycin sensitivity-conferring protein; Uh M et al.; The gene coding for the yeast Saccharomyces cerevisiae mitochondrial oligomycin sensitivity-conferring protein (OSCP) has been sequenced, and the gene products have been characterized . The OSCP is subunit 5 of the mitochondrial ATP synthase, a multimeric protein complex . As such, the gene coding for the yeast OSCP is referred to here as the ATP5 gene . From the predicted primary sequence, the calculated molecular weight of the immature yeast OSCP is 22,813 and the amino acid sequence is 35% identical and 65% homologous to bovine OSCP . A null mutant has been constructed . This mutant strain is unable to grow on glycerol medium, has no detectable oligomycin-sensitive ATPase activity, and has no detectable immune reactive proteins with the corresponding molecular weight of the OSCP (using antibodies reactive to the yeast OSCP) . The transcription products of the yeast gene have been characterized . There is a single major transcript from the ATP5 gene of 1.05 kilobases . The level of the transcription product is increased from 3-5-fold after growth in galactose medium as compared to cells grown in glucose medium . The transcriptional initiation sites were determined to occur at +68(G) and +69(T) at comparable frequency and were not dependent on the growth medium . These results suggest that transcription of the ATP5 gene is catabolite-repressed.

J Cell Physiol, 1990 Nov, 145(2), 253 - 61
The Na+,K+,2Cl-cotransport system in HeLa cells: aspects of its physiological regulation; Kort JJ et al.; We have previously reported on the biochemical properties of a Na+,K+,2Cl-cotransport system in HeLa cells and here we deal with aspects of its physiological regulation . Na+,K+,2Cl-cotransport in HeLa cells was studied by 86Rb+ influx and 86Rb+/22Na+ efflux measurements . The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on 86Rb+ flux, mediated by the bumetanide-sensitive Na+,K+,2Cl-cotransport system and the ouabain-sensitive Na+/K(+)-pump, were investigated . ANP reduced bumetanide-sensitive 86Rb+ influx under isotonic as well as under hypertonic conditions . Similar decrease of bumetanide-sensitive 86Rb+ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a beta-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive 86Rb+ influx . Furthermore, efflux of 86Rb+ and 22Na+ was greatly reduced in the presence of bumetanide and ANP . Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim . Biophys . Res . Commun . 168: 148-154, 1990), this study indicates that ANP participates in the regulation of the Na+,K+,2Cl-cotransport system in HeLa cells . Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative alpha-methyl-aminoisobutyric acid (metAIB, 1 and 5 mM, respectively) also reduced Na+,K+,2Cl-cotransport-mediated 86Rb+ uptake and diminished the stimulatory effect of hypertonicity on the contransporter . In addition, the Na+/K(+)-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-GMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na+/K(+)-pump.

Mol Cell Biol, 1990 Nov, 10(11), 5753 - 62
In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins; Nye SH et al.; To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region . When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion . Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain . A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer . Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS . Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting . Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space . By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.

New Biol, 1990 Nov, 2(11), 1034 - 43
Induction of nuclear NF-kappa B DNA binding activity after exposure of lymphoid cells to soluble tax1 protein; Lindholm PF et al.; We demonstrate that purified HTLV-I Tax1 protein can be taken up by 70Z/3 lymphoid cells and localized in both the nuclear and cytoplasmic compartments . Introduction of the Tax1 protein into the growth medium of 70Z/3 cells resulted in the rapid and transient induction of NF-kappa B binding activity in the nuclear fraction . Tax1 activation of NF-kappa B was not sensitive to either staurosporin or prolonged stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, suggesting that Tax1-dependent NF-kappa B activation did not require the protein kinase C pathway . Purified Tax1 did not directly increase NF-kappa B binding activity in 70Z/3 cytoplasmic extracts, suggesting that NF-kappa B induction may require cellular factors . Western blot and competitive radioimmunoassays demonstrated that Tax1 protein was present in the tissue culture media of HTLV-I-transformed cell lines . These results show that extracellular Tax1 may regulate cellular gene expression in noninfected cells.

Differentiation, 1990 Nov, 45(2), 84 - 95
Basement membrane components secreted by mouse yolk sac carcinoma cell lines; Damjanov A et al.; Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac carcinoma respectively . Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent . LRD was composed of a heterogeneous cell population and formed embryoid bodies . NE secreted soluble laminin, osteonectin, entactin and fibronectin but did not form visible pericellular matrix . ME formed pericellular matrix which was composed of laminin and entactin, but did not contain fibronectin . The LRD cells formed pericellular matrix which was composed of laminin, entactin and fibronectin . Whereas laminin from ME and LRD reacted with polyclonal antibodies and a monoclonal antibody to parietal yolk sac laminin, the laminin from NE cells was unreactive with the monoclonal antibody . Osteonectin was found in the supernatant of LRD and ME, but could not be demonstrated immunohistochemically in the extracellular matrix . We conclude that some extracellular matrix components, such as laminin and fibronectin, are produced not only by yolk sac carcinoma cells but by nullipotent EC as well, although the latter do not assemble them into extracellular matrix . Laminin produced by EC is immunochemically different from laminin secreted by yolk sac carcinoma . The extracellular matrix produced by mixed parieto-visceral yolk sac carcinoma is different from the matrix laid down by the pure PYS in that the latter does not contain fibronectin . The lack of osteonectin in the extracellular matrix of yolk sac carcinoma cells indicates that not all polypeptides secreted by these cell lines are incorporated into the extracellular matrix . The new cell lines described in this paper differ with regard to their capacity to form extracellular matrix and secrete its various components . Hence they could be used for further studies of basement membrane assembly in vitro.

Pigment Cell Res, 1990 Nov, 3(5), 243 - 50
Stimulation of cultured iridophores by amphibian ventral conditioned medium; Bagnara JT et al.; That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R . pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15 . Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R . pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period . Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation . Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P . dacnicolor in VCM . The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians . The results are discussed in terms of a prevailing theory about pigment cell origins and development.

Development, 1990 Nov, 110(3), 805 - 13
Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system; Brauer PR et al.; Basement membranes (BMs) are specialized extracellular matrices that have important roles in cell attachment, migration, growth and differentiation . The murine teratocarcinoma cell line, M1536-B3, has been shown to produce a model BM composed of laminin, entactin and heparan sulfate proteoglycans but lacking collagen . Therefore, M1536-B3 cells are an excellent model system in which to study the role of non-collagenous components in BM assembly . We have used these cells to test for a requirement of mature heparan sulfate (HS) chains in BM assembly . Growth of M1536-B3 cells in the presence of chlorate, an inhibitor of activated sulfate synthesis, resulted in a dose-dependent decrease in the sulfation of glycosaminoglycans and reduction in the charge density of the isolated HS . The undersulfated HS from chlorate-treated cells had a decreased binding capacity for laminin when compared with control HS . Concurrent with these changes in sulfation, chlorate treatment of M1536-B3 cells resulted in the failure of BM assembly, which was restored upon removal of the chlorate from the growth medium . These results were not due to major alterations in cell attachment, spreading, growth, protein synthesis, or to an inability of the cells to synthesize and secrete laminin . These data suggest that the sulfation of HS and its subsequent ability to interact with other BM components play major roles in the assembly and structure of BMs.

Mikrobiologiia, 1990 Nov-Dec, 59(6), 948 - 55
{Possible existence of alternative secretion routes of acid phosphatase in the yeast Saccharomyces cerevisiae}; Shnyreva MG et al.; The secretion of different acid phosphatase forms into the growth medium was studied in the yeast Saccharomyces cerevisiae . The secretion of constitutive acid phosphatase highly correlated with the process of bud formation . This exoenzyme might be involved in the construction of the bud cell wall . Analysis of the rate, at which repressible acid phosphatase was excreted into the growth medium, as well as a negative correlation between its secretion and the process of bud formation were indicative of an additional route via which the repressible form of acid phosphatase was secreted . It is possible that yeast cells contain vesicles similar to "safe" vesicles in higher eukaryotic cells.

J Cell Sci, 1990 Nov, 97 ( Pt 3), 473 - 8
Senescent human diploid fibroblasts are able to support DNA synthesis and to express markers associated with proliferation; Kill IR et al.; The characteristic limited reproductive life-span of normal human fibroblasts in culture is due to a steadily decreasing fraction of cells able to proliferate in the standard rich growth media . We have observed that restricting the growth factor supply to old cells for variable lengths of time in culture increases the fraction of cells that can enter S-phase; although these cells do not go on to divide . Thus, it seems that there is a transient phase between the proliferating state and the irreversibly post-mitotic, senescent state . Perhaps a 'quiescent-G0' state, which can be maintained in the presence of growth factors, is a stage on the pathway to mortalization and senescence.

Experientia, 1990 Oct 15, 46(10), 1032 - 7
Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells; Hoh YK et al.; Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner . {3H}Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-{4-(2-diethylaminoethoxy)phenyl}-1,2-diphenyl-2-chloroethylene) . The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol . Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth . Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001) . This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein . In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity . The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation . Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not . Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of {3H}tamoxifen (1-{4-(2-dimethylaminoethoxy)phenyl}-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory . Unsaturated fatty acids have previously been shown to inhibit binding of {3H}tamoxifen to the antiestrogen-binding protein in a cell-free system . The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.

Biotechnol Appl Biochem, 1990 Oct, 12(5), 567 - 78
Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli; Lundell D et al.; The bacterial membrane serves both as a cell organelle and as a barrier for segregating the metabolically active cytoplasm from the extracellular milieu . Thus we can use plasmid vectors designed to produce a hybrid protein containing an efficient signal peptide coupled to the amino terminus of the cloned heterologous protein (secretion cloning vectors) for the production of proteins which are insoluble, proteolytically sensitive, or bacteriocidal when produced in the cytoplasm of Escherichia coli . We demonstrate that human granulocyte-macrophage colony stimulating factor can be isolated as an active species only after transport into the bacterial periplasm . Production of the protein in the bacterial cytoplasm is bacteriocidal . We also demonstrate that biologically active human interleukin 4 appears only after transport of the protein into the bacterial growth medium . The protein forms membrane-associated aggregates in the cytoplasm, and demonstrates an active but nonnative conformation when expressed in the periplasm . This may correlate with the affinity of the interleukin 4 molecule for negatively charged macromolecules, including bacterial membrane components and bacterial lipopolysaccharides, which may alter the folding pathway inside the cell.

J Submicrosc Cytol Pathol, 1990 Oct, 22(4), 523 - 7
An ultrastructural evaluation of toluene toxicity using cultured mammalian cells; Mollenhauer HH et al.; Animal cells were cultured in media containing toluene and then examined by electron microscopy to determine changes in subcellular architecture . Cellular viability and cloning studies indicated toluene toxicity at concentrations of 50-500 ppm . At all dosage levels, toluene accelerated cell death compared to comparable control cells that had not been exposed to toluene . However, media composition and, particularly, the presence or absence of fetal bovine serum (FBS), markedly affected toluene toxicity . In media without FBS reversible changes in filopodia and cell shape were identified after 15 min of exposure to as little as 50 ppm toluene, and cell death occurred within 5 min at concentrations of 500 ppm toluene . In media with FBS, no filopodial changes were observed at 50 ppm toluene, and cell death, although accelerated, was clearly evident only after 24-48 h of exposure to 200-500 ppm toluene . Except for the cell-surface changes, chronic toluene exposures (50-100 ppm without FBS or 200-500 ppm with FBS in the growth medium) produced no unusual intracellular changes until 24-48 h of toluene exposure . With toluene exposures of 24 h or longer, cellular changes included condensation of heterochromatin in nuclei, formation of bulbous protuberances at the cell surface, loss of polyribosomes (but not ribosomes) and, ultimately, degeneration of organelles . These late changes were irreversible and the prelude to cell death.

Biophys J, 1990 Oct, 58(4), 919 - 30
Chemotaxis of bacteria in glass capillary arrays . Escherichia coli, motility, microchannel plate, and light scattering; Berg HC et al.; Random and directed motility of bacterial populations were assayed by monitoring the flux of bacteria through a microchannel plate (a porous glass plate comprising a fused array of capillary tubes) separating two identical stirred chambers . Cells, washed free of growth medium by a new filtration method, were added to one chamber at a low density . Their number in the other chamber was determined from the amount of light scattered from a beam of a laser diode and recorded on a strip chart . Diffusion coefficients were computed from fluxes observed in the absence of chemical gradients, and chemotaxis drift velocities were computed from fluxes observed in their presence . Cells migrated through tubes of diam 10 microns more rapidly than through tubes of diam 50 microns, suggesting that the straight segments of their tracks were aligned with the axes of the smaller tubes . Mutants that are motile but nonchemotactic could be selected because they move through the microchannel plate in the face of an adverse gradient . Weak chemotactic responses were assessed from ratios of fluxes observed in paired experiments in which the sign of the gradient of attractant was reversed . Studies were made of wild-type Escherichia coli and mutants that are nonmotile, tumblely, smooth-swimming, aspartate-blind, or defective in methylation and demethylation . Chemotaxis drift velocities for the latter mutants (cheRcheB) were quite small.

Mutat Res, 1990 Oct, 242(2), 163 - 8
Cytogenetic study for possible mutagenic activity induced by ice-nucleation bacteria or their metabolic products in human lymphocytes in vitro; Lialiaris T et al.; A means for eliminating ice-nucleation-active (INA) bacteria, the microorganisms responsible for frost damage to plants at mild freezing temperatures, is the use as competitors of other naturally occurring, non-nucleating strains . Inactive mutants (INA-) of INA bacteria have been produced by genetic or chemical methods and proposed for biological control of INA populations . Since, however, the application of these INA- mutants in the field may create health hazards to animals, we have studied the possible mutagenic activity of the INA- mutants by examining chromosome aberrations, sister-chromatid exchange (SCE) frequencies, and proliferation kinetics of human lymphocyte cultures . These cultures were treated with: (a) a naturally occurring INA- bacterium (p 767), (b) 2 parental strains (cit 7 and cit 13) of INA bacteria isolated from Citrus orchards, and (c) 2 INA- mutant strains (cit 7 del 1b and cit 13-12), produced, respectively, by chemical modification and by deletion of the corresponding parental strains . Neither whole bacteria nor infiltrates of bacterial growth media, in which toxic metabolic bacterial products might have been released, induced elevation of either chromosome aberrations and SCEs or a cell-division delay . Negative results were also obtained when sonicated bacteria were tested for possible intracellular mutagenic components.

Biochem J, 1990 Oct 1, 271(1), 31 - 6
Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line; Odum L et al.; A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene . Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells . Gel chromatography and reverse-phase h.p.l.c . revealed both sulphated and nonsulphated CCK-8 in the cells . The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK . Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold . The increase was first noted after 3 days of treatment . Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured . Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis . Finally, chloroquine inhibited the alpha-carboxyamidation of CCK . We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.

J Bacteriol, 1990 Oct, 172(10), 6142 - 4
The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system; Steiert PS et al.; The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex . Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity . A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant . Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels . The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity . Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.

Virology, 1990 Oct, 178(2), 401 - 9
Transformation of chicken fibroblasts by the v-fms oncogene; Tamura T et al.; The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membrane-associated tyrosine kinase (gp140v-fms) which is closely related, both structurally and functionally, to the c-fms-specified receptor for the macrophage colony stimulating factor (CSF-1) . In mammalian fibroblasts, the natural producers of CSF-1, expression of v-fms leads to cell transformation . To study the interaction between CSF-1 and gp140v-fms molecules in a cell system that does not produce endogenous cross-reactive CSF-1, we have expressed the entire v-fms gene as well as a nontransforming deletion mutant (SC2) in chicken embryo cells (CEC) . For this purpose the avian retroviral vectors pDS3 and pREP, based on Rous sarcoma virus, were used to isolate recombinant virus particles . CEC infected with virus that carried the entire v-fms gene expressed high amounts of gp140v-fms, comparable to those in SM-FeSV transformed NRK cells . However, these CEC remained flat, retained their fibronectin network, and did not produce enhanced levels of plasminogen activator . The cells grew faster than control CEC for more than 8 weeks but failed to form colonies in soft agar . Within 2 days after addition of CSF-1 to the growth medium, a transformed cell phenotype was induced, as judged by loss of the fibronectin network, again with a growth rate fourfold faster than that of the parental cells and with colony formation in soft agar . Moreover, human CSF-1 caused a rapid tyrosine phosphorylation of v-fms molecules detectable within 5 min after addition of the growth factor . In contrast, CSF-1 had none of the above effects on cells that expressed the SC2 v-fms deletion mutant.

Membr Biochem, 1990 Oct-Dec, 9(4), 263 - 77
Direct regulation of Na(+)-dependent myo-inositol transport by sugars in retinal pigment epithelium: role of phorbol ester and staurosporin; Khatami M et al.; An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32) . The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells . RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks . The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, {MI}i, to external free MI concentration, {MI}i/{MI}o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media . The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively . In addition, cellular capacity to bind to {3H}phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively . Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI . An 18 +/- 8% reduction in {3H}thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose . Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, {3H}phlorizin binding, cell number, and DNA synthesis . However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells . Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Plant Mol Biol, 1990 Oct, 15(4), 633 - 42
Copper-induced expression, cloning, and regulatory studies of the plastocyanin gene from the cyanobacterium Synechocystis sp . PCC 6803; Briggs LM et al.; Plastocyanin can be detected in Synechocystis sp . PCC 6803 when 3 microM copper is added to the growth medium, BG-11 . The plastocyanin gene (petE) was cloned from a genomic lambda EMBL 3 library by screening with the petE gene from Anabaena sp . PCC 7937 . The Synechocystis 6803 petE gene is present as a single copy and, as deduced from the DNA sequence, encodes a precursor protein of 126 amino acids . The predicted 29 amino acid transit peptide shows substantial homology to the Anabaena 7937 transit peptide, thought to direct the plastocyanin precursor to the thylakoid lumen . Putative promoter sites -16 and -38 base pairs from the start of the petE gene have been identified . The deduced amino acid sequence has the greatest homology (61%) to the green alga Scenedemus obliquus plastocyanin . Despite the lower homology, the copper binding residues and certain aromatic residues remain highly conserved . Northern hybridization analysis indicates that the Synechocystis sp . PCC 6803 petE gene is not transcriptionally regulated since the accumulation of petE mRNA appears to be independent of the copper concentration in the growth media . The possibility of an additional polypeptide needed to facilitate the electron transfer from plastocyanin to P700+ is also discussed.

J Invest Dermatol, 1990 Oct, 95(4), 441 - 5
Successful culture of adult human melanocytes obtained from normal and vitiligo donors; Medrano EE et al.; The development of growth conditions for human melanocytes from uninvolved skin from vitiligo patients and age-matched normal adults is a prerequisite to understanding the etiology of this pigmentary disorder . By using new growth conditions, pure melanocyte cultures were prepared from normal adults and the pigmented skin of vitiligo donors . Both cell types grew without a lag period and were maintained for more than six months (4-8 passages) . The cultures could be expanded from several thousand melanocytes in the original cell suspension to several million cells (2-7 x 10(6} in pure culture . To obtain these results, the current standard conditions for the culture of fetal melanocytes were substantially modified . MEM-S medium was less satisfactory than MCDB-153 . Adult normal and vitiligo cells also required the presence of exogenous catalase (20 micrograms/ml) during isolation and primary seeding . Thereafter, this enzyme was not necessary . Melanocytes grew best and gave the highest yields if the concentrations of both calcium (200 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) were low (4-8 nM) . Other growth factors included in the MCDB-153 media were bFGF, crude bovine pituitary extract, insulin, transferrin, hydrocortisone, 5% FCS, and the antioxidant alpha-tocopherol . Cholera toxin and isobutylmethylxanthine (IBMX) were omitted from the MCDB-153 growth medium because they slowed down growth even at very low concentrations . The results indicate adult and vitiligo melanocytes can be cultured . Preliminary studies of the normal and vitiligo cells indicate that vitiligo melanocytes retain some of the ultrastructural abnormalities observed in skin even when grown in culture.

Cancer Res, 1990 Oct 1, 50(19), 6396 - 404
Induction of cells with phenotypic features of neuronal cells by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate in a human parotid gland adenocarcinoma cell line in culture; Nagamine S et al.; A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP) . Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance . In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells . The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells . Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP . After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells . These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Biochim Biophys Acta, 1990 Sep 18, 1046(2), 144 - 50
Characterization and regulation of phosphatidylglycerolphosphate phosphatase in Saccharomyces cerevisiae; Kelly BL et al.; Phosphatidylglycerophosphatase (EC 3.1.3.27) activity was characterized in mitochondrial extracts from Saccharomyces cerevisiae . The enzyme has a pH optimum of 5.5 . Maximum activity occurs in the presence of Triton X-100 (5 mM) and cobalt or magnesium ions (5 mM) . The apparent Km for PGP is 14.6 microM . The temperature optimum is between 50 degrees C and 60 degrees C . The enzyme is labile above 50 degrees C . The presence of inositol in growth media results in a slight but reproducible increase in PGPase activity in mitochondrial extracts from glucose-grown cells but not glycerol-grown cells . The inositol effect is not seen in crude cell extracts . Carbon source does not affect PGPase activity in mitochondrial extracts or in crude cell extracts.

Biochem J, 1990 Sep 15, 270(3), 673 - 8
Riboflavin-dependent expression of flavoenzymes of the nicotine regulon of Arthrobacter oxidans; Brandsch R et al.; In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium . Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin . Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes . When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels . When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides . No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin . Pulse-chase experiments with {35S}methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration . The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown . Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells . From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

In Vitro Cell Dev Biol, 1990 Sep, 26(9), 871 - 7
Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: absence of autocrine effects; Guo YS et al.; We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells . The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown . For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells . The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of {3H}thymidine incorporation and cell number . A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum . The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells . Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of {3H}thymidine uptake by the cells, and a significant increase in cell number . The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells . The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself . To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed.

J Cell Physiol, 1990 Sep, 144(3), 492 - 7
Nerve growth factor and fibroblast growth factor influence post-fusion expression of Na-channels in cultured rat skeletal muscle; Brodie C et al.; We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)-sensitive Na-channels in cultured rat skeletal muscle . Measurements were made of specific binding of {3H}saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the physiological expression of Na-channel density . Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later . Both growth factors (GF) caused dose-related increases in Na-channels compared with myotubes maintained in normal, serum-supplemented growth medium . Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml . Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd . Similar increases occurred on rate of rise and frequency spontaneous action potential . Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX-binding induced by GF treatment . The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.

Genetika, 1990 Sep, 26(9), 1679 - 81
{Increase in the fraction of non-repaired mutations in Escherichia coli cells, incubated in the absence of glucose after UV-irradiation}; Filippov VD et al.; In E . coli WP2 trpE65 cells irradiated with UV-dose of 11 J/m2, the additional small portion of induced Trp+ mutations became resistant to photoreactivation or "dark" (excision) repair after a short-termed (10-30 min) postirradiation incubation of bacteria in a minimal medium deprived of glucose and tryptophan . Since protein synthesis could not proceed in those cells because of the lack of energy and tryptophan, the data indicate that an unknown mechanism exists which imparts some mutations with the resistance to antimutagenic repair in the absence of the inducible mutagenic system . In the light of this result, one could suggest that the normal process of mutation fixation (that is the loss of sensitivity of mutations to photoreactivation or to excision repair in cells incubated in growth medium after irradiation) should not necessarily be a direct consequence of manifestation of the activity of an inducible mutagenic system.

Poult Sci, 1990 Sep, 69(9), 1582 - 9
Effect of d-mannose on fimbriae of bacterial isolates from chicken carcasses; deGraft-Hanson J et al.; Pseudomonad-like microbial isolates were obtained from commercially processed broiler chicken carcasses for use in experiments to determine if d-mannose would interfere with their ability to form pellicles and rims and prevent agglutination of blood and yeast cells . Inhibition of agglutination, rim formation, and pellicle formation would provide evidence that d-mannose acts on the fimbriae to prevent attachment of microorganisms to surfaces . Presence of 4% d-mannose in the growth medium interfered with the formation and stability of the pellicle in some isolates . The pellicles affected by d-mannose did not cover the entire surface and were easily dispersed . Addition of d-mannose did not prevent the formation of rims to the same extent it prevented pellicle formation, and it was concluded that d-mannose did not completely prevent growth of fimbriae . The addition of d-mannose prevented the agglutination of 1% sheep blood, 1% horse blood, and yeast cells by interfering with the formation of fimbriae or the mechanism of attachment by fimbriae . These experiments provided evidence that d-mannose can be used to prevent attachment of isolates normally found on chicken carcasses to specific cell surfaces by occupying mannose-sensitive receptor sites on the cell.

Biotechnol Prog, 1990 Sep-Oct, 6(5), 383 - 90
Protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress; Goldblum S et al.; The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), was evaluated . TN-368 and SF-9 cells were found to be equally sensitive to laminar shear stress . Methylcellulose {0.5% (w/v) Dow E4M Methocel} and dextran {4.5% (w/v)} increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives . It was observed that the protective effect of Pluronic F-68 was concentration-dependent: 0.2% and 0.3% (w/v) F-68 increased the resistance of SF-9 cells to shear stress by factors of 15 and 42, respectively . However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v) . F-68 at 0.2% only increased the resistance of TN-368 cells by a factor of 6 . It is believed that the protection is a result of the polymer adsorbing to the cell membrane . None of the polymer additives tested had a significant effect on SF-9 or TN-368 growth rate.

Cytotechnology, 1990 Sep, 4(2), 111 - 9
Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells; Sambanis A et al.; Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3-0.5 ng/10(5) cells-hour when exposed to medium without secretagogues . When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10(5) cells-hour while intracellular hGH declined sharply . Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial . In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted . When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles . The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur . Operational modifications that may improve the performance of cycling schemes are discussed.

J Biol Chem, 1990 Aug 15, 265(23), 13641 - 9
Toxicity and effects of epidermal growth factor on glucose metabolism of MDA-468 human breast cancer cells; Kaplan O et al.; Epidermal growth factor (EGF) has an in vitro inhibitory effect on tumor cells which exhibit a high number of EGF receptors (EGFR) . Studies were performed in order to delineate the effects of EGF on glucose metabolism of MDA-468 human breast cancer cells, which have a large number of EGFR . Glucose consumption and lactate production were found to be substantially increased in MDA-468 cells following EGF exposure, while no such effects were detected in MCF-7 breast cancer cells, which have a very low number of EGFR . When glucose levels in the growth medium were increased, the toxicity of EGF was diminished . The energetic status of MDA-468 cells perfused with growth medium containing EGF was monitored by 31P magnetic resonance spectroscopy, and no signs of compromised metabolic state or viability were noted for up to 36 h . The rate of glucose transport and phosphorylation was quantitated by 13C magnetic resonance spectroscopy, utilizing {6-13C}2-deoxyglucose, and a 97% increase was found in MDA-468 cells following EGF administration . The profound effects of EGF on glucose metabolism in cells with very high numbers of EGFR and the lack of toxicity in the perfused system may indicate that the growth-inhibitory effect is confined to the in vitro cultured cells.

Science, 1990 Aug 3, 249(4968), 540 - 2
Regulation of activity of a transcriptional anti-terminator in E . coli by phosphorylation in vivo; Amster-Choder O et al.; Expression of the bgl operon of Escherichia coli is regulated in vitro by phosphorylation and dephosphorylation of a positive regulatory protein, BglG, which functions in its nonphosphorylated state as a transcriptional antiterminator . The degree of phosphorylation of BglG in vivo was shown to be dependent on the cellular levels of BglF protein, which is both the BglG kinase and phosphatase . The degree of phosphorylation of BglG also depended on the presence or absence of a beta-glucoside, the inducer of operon expression . Addition of inducer to cells in growth medium resulted in rapid dephosphorylation of phosphorylated BglG . The bgl operon is thus regulated by a sensory system that modulates gene expression by protein phosphorylation and dephosphorylation in response to the external levels of inducer.

Exp Eye Res, 1990 Aug, 51(2), 119 - 29
The growth and behaviour of rat retinal Müller cells in vitro . 1 . An improved method for isolation and culture; Hicks D et al.; Eyeballs were enucleated from young (postnatal day 8-12) pigmented rats and the retinas were dissected free after soaking the globes overnight in growth medium . The retinas were digested with enzymes, dissociated and maintained in stationary culture in 10% serum supplemented growth medium . Cultures displayed extensive cellular outgrowth after 1-5 days, with abundant fusiform and epithelioid cells . Removal of aggregates and cellular debris after 6-7 days yielded a purified flat cell preparation, which could be maintained either as a primary culture for several weeks or passaged repeatedly as rapidly proliferating epithelioid cells . Staining with monoclonal antibodies RET-G1, G3 and G7, and polyclonal S-100, glutamine synthetase and carbonic acid anhydrase antisera, all markers for Muller cells, showed positive labelling of all cells present in these purified cultures, both primary and passaged cells . This contrasted with the use of RET-G2, anti Factor VIII and anti glial fibrillary acidic protein (GFAP) antibodies . RET-G2, another Muller cell marker, failed to recognize passaged cells . Anti Factor VIII also did not label any cells, and anti GFAP stained very few cells: these remained associated with aggregated material so that vigorous washing to remove loosely adherent tissue from primary cultures resulted in the total absence of GFAP positive cells . In addition, no GFAP positive cells were detected in passaged cells or in cells regrown following freezing and storage . The Muller cell nature of these flat cells in soaked retinal cultures was further supported by the specific uptake of 5-bromo-deoxyuridine by nuclei located within the inner nuclear layer of retinal fragments in vitro . Hence the soaking treatment greatly reduces the number of surviving astrocytes whilst stimulating the rapid growth of cells expressing many properties of mature retinal Muller cells.

Protein Eng, 1990 Aug, 3(8), 709 - 12
Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression; Murzina NV et al.; Three different forms of dihydrofolate reductase (DHFR) from Escherichia coli with amino acid replacements Thr35----Asp, Asn37----Ser and Arg57----His, and one form containing all three of these changes were obtained by oligonucleotide-directed mutagenesis . These amino acids are on the surface of the protein and two of them (Thr35 and Arg57) are invariant for known sequences of DHFR . Conversion of Asn37----Ser has no effect on the functional activity or the protein level in the cells . The Thr35----Asp replacement leads to a sharp decrease in the protein level, while the addition of a DHFR inhibitor, trimethoprim (Tmp), to the growth medium increases the level of DHFR in the cells . There is a very small quantity of DHFR with all three amino acid changes . The addition of Tmp to the growth medium also leads to an increase in the mutant protein levels . The mutant with the Arg57----His replacement renders the cells sensitive to Tmp, but the level of DHFR is the same as for the wild-type protein . It is suggested that the invariant Thr35 is important for the stable conformation of DHFR whereas Arg57 is essential for protein activity . Various structural and functional aspects of these results are discussed.

Am J Physiol, 1990 Aug, 259(2 Pt 1), L95 - 101
Stimulation of prostaglandin synthesis by hyperoxia in perinatal rat lung cells; Lee DS et al.; Prostaglandins (PGs) have been implicated in the development of pulmonary oxygen toxicity . We tested the hypothesis that hyperoxia modulates PG synthesis in a differentiation-arrested primary lung cell culture model in the rat at three developmental ages: day-20 gestation (term = 22 days), days 1 and 3 after birth . The time courses of the response to hyperoxia were defined in preconfluent lung cells as well as in growth-arrested, confluent cells . From days 4-8 after plating in growth medium containing 10% carbonstripped fetal bovine serum, exposure to 95% O2, in contrast to 1% O2, inhibited cell proliferation but significantly enhanced the production of PGI2 and, to a lesser extent, PGE2 at all three ages . The capacity to metabolize exogenous arachidonic acid (AA) to PGI2 was also increased two-to threefold (P less than 0.01) . Cellular release of lactate dehydrogenase, a measure of O2 toxicity, remained unchanged during exposure to 1% O2 but increased fivefold between 48 and 96 h after exposure to hyperoxia (from 2% total to 10.5%, P less than 0.01) . In confluent, growtharrested cells, under serum-free conditions, exposure to hyperoxia for 24-48 h resulted in a similar induction of PG synthesis . Our results suggest that hyperoxia stimulates PG synthesis in the perinatal rat lung and that this effect is independent of cell growth or the presence of serum . We speculate that this hyperoxia-induced PG synthesis is a relatively early response to oxidant stress and may serve as an useful early marker for O2 toxicity in perinatal lung cells.

Cancer Res, 1990 Jul 15, 50(14), 4199 - 203
Reversal of the multidrug resistance phenotype with cremophor EL, a common vehicle for water-insoluble vitamins and drugs; Woodcock DM et al.; A polyethoxylated castor oil, Cremophor EL, which is used as a vehicle for p.o . and i.v . administration of water-insoluble compounds in humans, can reverse the multidrug resistance (MDR) phenotype at doses which are likely to be readily achievable clinically . Using flow cytofluorometric analysis of daunorubicin (DNR) uptake as a measure of the expression of the MDR phenotype, Cremophor EL (1:10(3} in the growth medium increased intracellular DNR in an MDR cell line, R100 cells, to levels similar to that observed in the drug-sensitive parental cells, CCRF-CEM . A similar Cremophor EL-induced increase in DNR uptake was also observed in an unrelated MDR cell line derived from K562 cells . Cremophor EL (less than or equal to 3:10(4} did not inhibit the growth of CCRF-CEM cells or its vinblastine-resistant derivative, R100 cells, but would significantly increase the sensitivity of R100 cells to both vinblastine and DNR . Also Cremophor EL did not increase the sensitivity of normal bone marrow progenitor cells cultured in vitro to high concentrations of vinblastine . Cremophor EL may prove to be a relatively pharmacologically inactive addition to chemotherapeutic protocols which may be able to reverse the MDR phenotype in tumors and also help to prevent the selection of MDR cell variants from within a tumor cell population during chemotherapy.

Int J Cancer, 1990 Jul 15, 46(1), 56 - 60
Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity; Tsuboi R et al.; Three human tumor cell lines, Bowes' melanoma, HT1080 and Osmond cells, were characterized for their ability to invade the amniotic membrane and their production of plasminogen activator . Bowes' melanoma cells, which release large amounts of tissue plasminogen activator (tPA), were poorly invasive on the amniotic membrane . The addition of plasmin inhibitors, anti-tPA antibody or tissue inhibitor of metalloproteinase (TIMP) to the amnion assay enhanced invasiveness . The depletion of plasminogen from the growth medium also enhanced the degree of invasiveness . Similarly, HT1080 cells, which produce high levels of urokinase-type plasminogen activator (uPA), were poorly invasive under standard conditions but invasion was enhanced by plasmin inhibitors or anti-uPA antibodies . Conversely, Osmond cells, which produce low levels of uPA, were very invasive on the amniotic membrane . Invasion by these cells was blocked by the addition of plasmin inhibitors or anti-uPA antibodies to the amnion assay . These results suggest that invasion requires only a minimum level of PA activity and that, as PA production exceeds this optimal level, the degree of invasion decreases . We propose that high levels of plasmin, generated by the tPA or uPA secreted by the cells, may cause uncontrolled matrix degradation and interrupt the interaction of cells and matrix in the early stages of invasion . The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts and result in an enhancement of invasion.

J Biol Chem, 1990 Jul 15, 265(20), 11605 - 14
Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli; Hernandez VJ et al.; We have fused the ribosomal RNA promoter P1 from the rrnB gene of Escherichia coli to lacZ and examined its guanosine tetraphosphate (ppGpp)-dependent expression at different growth rates . The rrnB P1-lacZ fusion was constructed on plasmid vectors and then recombined into the chromosome of a delta lac relA1 strain close to the normal location of the rrnB locus and in the normal orientation, coincident with the direction of replication . A series of spoT strains differing in the severity of their SpoT defect were analyzed with respect to their growth characteristics and ppGpp metabolism . The spoT203 allele was introduced into the rrnB P1-lacZ fusion containing strain and used to manipulate the ppGpp concentration independently of the growth medium . 1) The levels of ppGpp during exponential growth are decreased in rich media due to a decreased activity of (p)ppGpp synthetase II (PSII) . 2) The specific activity of rrn P1-directed beta-galactosidase was increased in a parabolic fashion with increasing growth rate . A theoretical analysis showed that this was to be expected since enzyme expression from a stringently controlled promoter is affected by changes in the growth rate both via the control of the promoter, and indirectly via the control of bulk mRNA synthesis . 3) The observer specific enzyme activities were converted into rrnB P1 promoter activities, given as lacZ transcription relative to the total rate of transcription . The rrn P1 promoter activity decreased exponentially with increasing cytoplasmic concentration of ppGpp, independent of whether a given level of ppGpp was achieved by using different growth media or by using a spoT allele . These results support two hypotheses: (i) that RNA polymerase is partitioned by ppGpp into two fractions with different abilities to initiate transcription at rrn P1 promoters; and (ii) that during exponential growth ppGpp is derived from ppGpp synthetase II (PSII) . Together these hypotheses predict that the growth rate control of rRNA synthesis reflects a control of PSII activity which is regulated by the composition of the growth medium.

Neurochem Res, 1990 Jul, 15(7), 751 - 4
Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells; Tholey G et al.; The effects of physiological concentrations of K+ on Mn2+ accumulation were compared in rat glial cells and neurons in culture . Increasing the K+ concentration in growth medium increased significantly the Mn2+ level of the cultivated cells, with glial cells more affected than neurons . Ethanol markedly increased the Mn2+ accumulation within glia but not within neurons while ouabain caused inhibition of Mn2+ uptake with neurons and glial cells . A modulation of the total protein synthesis by Mn2+ and ethanol level in the growth medium was observed with glial cells . These data suggest that the mechanisms involved in Mn2+ accumulation in glial cells are different from those present in neurons . Moreover, the results are consistent with the hypothesis that Mn2+ plays a regulatory role in glial cell metabolism.

In Vitro Cell Dev Biol, 1990 Jul, 26(7), 691 - 700
Cultivation of HeLa cells with fetal bovine serum or Ultroser G: effects on the plasma membrane constitution; Blixt Y et al.; Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis . Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G- supplemented cells, respectively . Plasma membranes from cells grown with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells . The former membranes also showed a 3 times higher specific {3H}acetate labeling of cholesterol, indicating a higher de novo synthesis of cholesterol . Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings . Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells . However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe.

Proc Natl Acad Sci U S A, 1990 Jul, 87(14), 5405 - 9
Developmental repression of growth and gene expression in Aspergillus; Adams TH et al.; Asexual reproductive development can be initiated in Aspergillus nidulans in the presence of excess nutrients through artificial induction of the developmental regulatory genes brlA or abaA by fusing the genes to the promoter from the alcohol dehydrogenase I gene (alcA) and culturing cells in the presence of an inducing alcohol . Artificially induced development completely inhibits growth and represses expression of the endogenous alcA gene and the coordinately controlled aldehyde dehydrogenase gene (aldA) . Repression of alcA and aldA expression probably occurs at both the transcriptional and posttranslational levels . We propose that developmental induction results in a generalized metabolic shutdown, leading to an inability of cells to acquire nutrients from the growth medium . Self-imposed nutrient limitation could reinforce the primary developmental stimulus and ensure progression through the asexual reproductive pathway.

Anticancer Res, 1990 Jul-Aug, 10(4), 1055 - 9
Gamma linolenic acid alters the cytotoxic activity of anticancer drugs on cultured human neuroblastoma cells; Ikushima S et al.; Gamma linolenic acid (GLA) by itself shows anti-tumor activity on various neoplastic cells in culture . We investigated the combined effect of GLA and anticancer drugs on two human neuroblastoma cell lines . The cytotoxic effect of Vinca alkaloids such as vincristine (VCR), vindesine (VDS) and vinblastine (VBL) was about 2-fold enhanced when GLA was added simultaneously to the growth medium . On the other hand, that of platinating agents such as cisplatin (CDDP) and carboplatin (CBDCA) was inhibited by GLA supplementation . The cytotoxic activity of other anticancer agents was not affected by GLA . Pharmacokinetic studies on VCR and CDDP demonstrated that intracellular accumulation of {3H}-VCR was about 1.5-fold increased in the cells pretreated by GLA, while, on the contrary, that of CDDP was rather decreased; cellular efflux of either drug was not affected . Malon dialdehyde (MDA) formation induced by supplemented GLA was not influenced by either VCR or CDDP . These results indicate that GLA exerts differential effects on the kinetics of anticancer drugs.

Zentralbl Veterinarmed B, 1990 Jul, 37(5), 337 - 44
Effect of frequent milkings on milk NAGase, plasmin, trypsin inhibitory capacity and the quality of whey as the growth medium for mastitis pathogens; Kaartinen L et al.; Unequal milking intervals affected milk somatic cell count and N-acetyl-beta-D-glucosaminidase (NAGase) activity . The total daily output of milk NAGase and plasmin decreased, if quarters were emptied frequently during the day . Mastitis pathogens showed stimulated growth in whey prepared from filled quarters as compared with growth in whey from quarters emptied frequently during the day . The quality of whey as growth medium for mastitis pathogens paralleled plasmin activity in respective milk samples . Adaptation of mastitis pathogens to grow in whey had an enhancing effect on bacterial growth during subsequent inoculations in whey . Bacteria probably "learn" to overcome the effect from endogenous antibacterial factors and to use nutrients present in whey.

Arch Biochem Biophys, 1990 Jul, 280(1), 159 - 66
Changes of some physical properties of isolated and purified plasma and thylakoid membrane vesicles from the freshwater cyanobacterium Synechococcus 6301 (Anacystis nidulans) during adaptation to salinity; Riviere ME et al.; Photoautotrophically growing cultures of the freshwater cyanobacterium Anacystis nidulans (Synechococcus sp.) became adapted to the presence of 0.4-0.5 M NaCl in the growth medium (about seawater level) with a lag phase of 2 days after which time the growth rate resumed at 80-90% of the control . Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process . Plasma and thylakoid membranes were separated from crude cell-free extracts of French pressure cell-treated Anacystis by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients . Concentrations of copper, iron, calcium, and magnesium ions were determined by inductively coupled plasma atomic emission spectrometry with EDTA-washed and dialyzed membrane preparations; salt adaptation was found to increase (decrease) the concentration of membrane-bound calcium in plasma (thylakoid) membranes, qualitatively reciprocal results being obtained for magnesium . Levels of plasma membrane-bound copper and iron roughly tripled during the adaptation process; by contrast, corresponding effects on thylakoid membranes were negligible . The size of the membrane vesicles was measured by quasi-elastic laser light-scattering and the electric surface charge of the membranes was measured by laser Doppler velocimetry . Salt adaptation decreased the mean diameter of plasma membrane vesicles to a much higher extent than that of thylakoid membrane vesicles . Overall surface charge densities of resting vesicles were only slightly affected by the salt treatment as was also seen from titration of the electrophoretic mobility of the vesicles with electrolytes . Yet, induction of (photosynthetic or respiratory) electron transport provoked a charge separation across the membrane which was easily measurable in terms of electrophoretic mobility . The results will be discussed with particular emphasis on the stimulated cytochrome c oxidase activity of plasma (but not thylakoid) membranes from salt-adapted cells compared to control cells and also with respect to the decreased ion permeability of the plasma membrane of salt grown cells.

Lab Invest, 1990 Jul, 63(1), 115 - 22
Growth of microvessels in serum-free matrix culture of rat aorta . A quantitative assay of angiogenesis in vitro; Nicosia RF et al.; We report here that rings of rat aorta embedded in gels of fibrin or collagen and cultured in MCDB 131, an optimized growth medium for microvascular endothelial cells, generate branching microvessels in the absence of serum or other soluble protein supplements . The angiogenic response is self-limited and can be quantitated by counting the newly formed microvessels daily in the living cultures . The microvascular growth curves are characteristic for each gel . Growth of microvessels in collagen gel peaks at the end of the 1st week and is followed by a rapid regression in the 2nd week . Fibrin gels, as compared with collagen, stimulate angiogenesis by 170%, support growth during the 2nd week, and protect the newly formed microvessels from early regression . Angiogenesis is inhibited by adding hydrocortisone to the culture medium . Conversely, a 230% stimulation of angiogenesis is obtained when aortic rings are cultured in collagen gels floating in serum-free medium conditioned by sarcoma 180 cells . Our results demonstrate that: (a) angiogenesis can be obtained reproducibly in serum-free culture; (b) serum-free culture is a sensitive method for testing the inhibitory or stimulatory effects of soluble or matrix factors on angiogenesis; (c) the aortic ring model can be used as a quantitative assay for the study of angiogenesis under chemically defined culture conditions.

Cancer Lett, 1990 Jun 30, 52(1), 83 - 9
Antiproliferative action of retinoic acid in cultured human brain tumour cells Gl-As-14(S); Mukherjee P et al.; We have employed human Gl-As-14(S) brain tumour cell line to study antiproliferative action of retinoic acid (RA) . RA (20 microM) caused a time-dependent, dose-dependent, cell seeding density-dependent reduction in cell proliferation in liquid medium and inhibited growth in agar . Growth inhibitory effects of RA were also affected by the concentration of fetal bovine serum (PBS) in the medium . All these effects could be reversed within 48 h after removal of RA from the growth medium . RA-treated cells also displayed reduced concanavalin A (Con A) binding ability by microhemadsorption technique . The results demonstrated that RA can suppress in this tumour cell line the expression of some properties frequently associated with transformed cells.

J Biol Chem, 1990 Jun 25, 265(18), 10541 - 50
A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments; Lewis MJ et al.; The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique . Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis . Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate . The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium . Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase . The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm . For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini . Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270 . These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation . These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation . Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.

J Natl Cancer Inst, 1990 Jun 20, 82(12), 1055 - 61
New carbon dioxide-independent basal growth medium for culture of diverse tumor and nontumor cells of human and nonhuman origin; Vistica DT et al.; A eukaryotic growth medium (Program Development Research Group Basal Growth Medium) was developed for CO2-independent maintenance and propagation of human and nonhuman tumor cell lines representing diverse histologies (e.g., cancers of the brain, colon, lung, ovary, and kidney, as well as leukemia and melanoma) . It was also shown to be suitable for the maintenance and propagation of nontumor cells of human and nonhuman derivation . The medium derives its buffering capacity primarily from beta-glycerophosphate, exhibits a stable physiologic pH of 7.3-7.4, and is optimized to facilitate growth in atmospheric CO2 . It is also useful in cellular growth and cytotoxicity assays based on either the metabolic reduction of tetrazolium reagents or protein staining . The 50% inhibitory concentration values obtained with carmustine, doxorubicin, and tamoxifen in cell lines maintained in the new medium under atmospheric CO2 were closely comparable to those obtained with these drugs against cells maintained in RPMI-1640 under a 5% CO2 environment.

Gene, 1990 Jun 15, 90(2), 255 - 62
Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae; Gabrielsen OS et al.; A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha) . Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml) . More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides . Three internal cleavage sites were identified in the hPTH molecule . The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends . The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material) . The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Gene, 1990 Jun 15, 90(2), 181 - 92
Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans; Johnstone IL et al.; Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis . This gene cluster is required for the assimilation of nitrate in A . nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively . The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed . Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively . The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium . Two types of transcripts for niiA were observed.

Cancer Res, 1990 Jun 15, 50(12), 3574 - 8
Interaction of methotrexate polyglutamates and dihydrofolate during leucovorin rescue in a human breast cancer cell line (MCF-7); Allegra CJ et al.; Previous investigations have suggested that high-dose methotrexate with leucovorin rescue is a potentially useful strategy for overcoming antifolate resistance . Interactions between methotrexate (MTX) and leucovorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reductase (MTX/MTX polyglutamates versus dihydrofolate) and other folate-dependent enzymes (MTX polyglutamates versus reduced folate substrates) . The present studies were designed to test the ability of dihydrofolate to compete with methotrexate and methotrexate polyglutamates for dihydrofolate reductase activity using an intact human breast carcinoma cell line (MCF-7) as the model system . Exposure of the breast cells to methotrexate for 24 h resulted in a concentration-dependent formation of methotrexate polyglutamates that markedly exceeded the dihydrofolate reductase-binding capacity for up to 24 h after the removal of drug from the growth media . Under these conditions of dihydrofolate reductase inhibition, we found that tritium-labeled dihydrofolate was capable of competing with methotrexate and its metabolites for dihydrofolate reductase activity as evidenced by the appearance of tritium-labeled reduced folates in the treated cells . We found the interaction between dihydrofolate and methotrexate to be dependent on the exposure concentrations of both methotrexate and dihydrofolate . These studies provide direct evidence that competition during leucovorin rescue occurs at the level of dihydrofolate reductase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.

Int J Dev Biol, 1990 Jun, 34(2), 319 - 22
Light-driven diurnal zonation in the filamentous fungus Fusarium solani; Das J et al.; Zonation in growing mycelia of Fusarium solani was induced by diurnal light/dark cycles . Only those parts of the hyphae that grew in darkness for less than 20 hours developed a zone of conidia after illumination . In continuous darkness, in continuous illumination, or after a transition from light to darkness, a conidiation zone failed to appear . Only light periods exceeding a few seconds but lasting less than 21 hours during a 24 hour light/dark cycle induced zonation . This zonation was not caused by periodic staling of the growth medium.

Lipids, 1990 Jun, 25(6), 311 - 5
Modification of phospholipid polar head group with monomethylethanolamine and dimethylethanolamine decreases cholesteryl ester and triacylglycerol synthesis in cultured human fibroblasts; Maziere C et al.; Modification of the phospholipid polar head group was achieved by supplementation of the growth medium of cultured human fibroblasts with the choline analogues monomethylethanolamine (ME) or dimethylethanolamine (DE) at a concentration of 80-200 micrograms/mL for 48 hr . The maximum concentration of phosphatidylmonomethylethanolamine (PME) or phosphatidyldimethylethanolamine (PDE) reached without affecting the phospholipid/protein ratio was about 45% of total phospholipids . Incorporation of oleic acid into cholesteryl esters and triacylglycerols was markedly inhibited after supplementation with ME or DE, and accounted for 60% and 40% of controls, respectively, at 200 micrograms/mL, whereas incorporation into phospholipids was not affected . AcylCoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) activities measured on cell-free extracts appeared to be decreased also by phospholipid polar head group modification, whereas the overall phospholipid acyltransferase activity remained unchanged . The intracellular content of cholesteryl esters and triacylglycerols, determined by the isotopic equilibrium method with radioactive cholesterol and glycerol, was found to be diminished to 50-60% and 40-50% of controls, respectively, after supplementation with the choline analogues . The study showed that modification of the phospholipid polar head group affects the activity of membrane-bound enzymes involved in the metabolism of neutral lipids.

In Vitro Cell Dev Biol, 1990 Jun, 26(6), 589 - 95
Human oral epithelial cell culture I . Improved conditions for reproducible culture in serum-free medium; Oda D et al.; Gingival tissue from healthy adult human donors was used as a source of epithelial cells for culture . An overnight incubation of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective tissue . This step minimized culture contamination with fibroblasts . The epithelium was then trypsinized to prepare a single cell suspension . The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated at 37 degrees C and 5% CO2 in a humid atmosphere . Primary cultures grew in small islands that coalesced at confluency . Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins of stratified squamous epithelium . Ultrastructurally, the cells contained distinct intermediate filaments . When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes . However, when cells were grown under physiologic calcium (1.2 mM), desmosomes were prominent and well developed . Cells were maintained in culture for over 100 d (7 passages).

Am J Pathol, 1990 Jun, 136(6), 1275 - 81
Retinoic acid stimulation of human dermal fibroblast proliferation is dependent on suboptimal extracellular Ca2+ concentration; Varani J et al.; Human dermal fibroblasts failed to proliferate when cultured in medium containing 0.15 mmol/l (millimolar) Ca2+ (keratinocyte growth medium {KGM}) but did when the external Ca2+ concentration was raised to 1.4 mmol/l . All-trans retinoic acid (retinoic acid) stimulated proliferation in KGM but did not further stimulate growth in Ca2(+)-supplemented KGM . The ability of retinoic acid to stimulate proliferation was inhibited in KGM prepared without Ca2+ or prepared with 0.03 mmol/l Ca2+ and in KGM treated with 1 mmol/l ethylene-glycol-bis-(beta-aminoethyl ether)N,N'-tetra acetic acid . Using 45Ca2+ to measure Ca2+ influx and efflux, it was found that retinoic acid minimally increased Ca2+ uptake into fibroblasts . In contrast, retinoic acid treatment of fibroblasts that had been pre-equilibrated for 1 day with 45Ca2+ inhibited release of intracellular Ca2+ into the extracellular fluid . Retinoic acid also stimulated 35S-methionine incorporation into trichloroacetic acid-precipitable material but in contrast to its effect on proliferation, stimulation of 35S-methionine incorporation occurred in both high-Ca2+ and low-Ca2+ medium . These data indicate that retinoic acid stimulation of proliferation, but not protein synthesis, is dependent on the concentration of Ca2+ in the extracellular environment.

J Cell Sci, 1990 Jun, 96 ( Pt 2), 263 - 70
Effects of thrombospondin antibody on the recovery of endothelial cells from hyperthermia; Ketis NV et al.; In addition to the increased synthesis of the classical heat-shock proteins (28,000, 71,000, 73,000, 90,000 and 100,000 Mr polypeptides) there is also an increase of thrombospondin in the growth medium of endothelial cells exposed to hyperthermia . The effect of a monoclonal antibody to thrombospondin on the recovery of endothelial cells from hyperthermia as it relates to cytoskeletal organization and cell spreading was assessed . The antibody interacts with the heparin-binding domain of thrombospondin in the extracellular matrix of cells . We report that during recovery from thermal insult at 37 degrees C, intermediate filaments, stress fibres and microtubules show distinct time-recovery characteristics in bovine aortic endothelial cells; that in the presence of this antibody the cytoskeleton is notably altered; that this antibody causes retraction of endothelial cell processes; and that the recovery of the cytoskeleton in endothelial cells exposed to hyperthermia is prevented by the thrombospondin antibody in the time frame examined . Our data suggest that the recovery of cells from heat shock requires the integrity of thrombospondin and its interactions.

Curr Genet, 1990 Jun, 17(6), 507 - 13
Mitochondrial modifications in a single nuclear mutant of Saccharomyces cerevisiae affected in cAMP-dependent protein phosphorylation; Dupont CH et al.; This paper reports studies of bioenergetic modifications in a TTR1 single-nuclear mutant, isolated as resistant to triethyltin, an inhibitor of mitochondrial ATPase, and effective in cAMP-dependent protein phosphorylation . This mutant appears to have lost the wild-type cell ability to respond to a decrease of oxygen concentration in the growth medium by a decrease of cytochrome concentration in the cell . ATP synthesis rate in mutant cells in both the prestationary and stationary phase of growth appeared increased in comparison to wild-type cells, as too was respiration rate . A comparative study of mitochondria extracted from wild-type and from TTR1 mutant cells showed an increase in respiration rate, an increase in ATP synthesis rate, and an increase in TPP+ uptake in mutant mitochondria . The specific ATPase activity, as well as its sensitivity to TET, appears to be similar for mitochondria extracted from both strains . It was proposed that the modification of mitochondrial biogenesis in the TTR1 mutant may be due to a response of the cell to an increase in ATP hydrolysis caused by the mutation . It is also possible that the modification in cAMP-dependent protein kinase regulation which appeared to occur in this mutant affects protein(s) involved in mitochondrial biogenesis.

J Bacteriol, 1990 Jun, 172(6), 3473 - 7
In vivo phosphorylation of OmpR, the transcription activator of the ompF and ompC genes in Escherichia coli; Forst S et al.; An in vivo approach was taken to assess whether the phosphorylated state of the transcription activator OmpR was affected by changes in the osmolarity of the growth medium or by mutations in envZ, the gene encoding the inner membrane histidine kinase that phosphorylates OmpR . We present results that support the view that increased phosphorylation of OmpR is correlated with enhanced expression of ompC . The in vivo phosphorylation approach was also used to show that OmpR can be phosphorylated in an envZ null strain . This result indicates that phosphorylation cross talk can occur in vivo between OmpR and a kinase(s) that is functionally homologous to envZ.

J Dent Res, 1990 Jun, 69(6), 1283 - 6
The possible role of oral epithelial cells in tissue-type plasminogen activator-related fibrinolysis in human saliva; Sindet-Pedersen S et al.; We studied the fibrinolytic activities of the following subfractions of unstimulated human whole saliva on plasminogen-rich fibrin plates: (1) submandibular saliva, (2) parotid saliva, and (3) smears of buccal epithelial cells from ten healthy males . A cell-bound plasminogen activator could be demonstrated in the sediments of all three subfractions of whole saliva . The incorporation of antibodies (goat IgG) against human two-chain tissue-type plasminogen activator (t-PA) could quench the assessed fibrinolytic activities, whereas additional experiments suggested the absence of urokinase-like and F XII-dependent activators of fibrinolysis . The determinations in growth medium from buccal-epithelial cell culture of t-PA antigen by means of enzyme-linked immunosorbent assay showed the presence of t-PA . These clinical and experimental findings suggest that buccal-epithelial cells produce t-PA, while the activity of t-PA in parotid and submandibular saliva is very low.

In Vitro Cell Dev Biol, 1990 Jun, 26(6), 596 - 603
Human oral epithelial cell culture . II . Keratin expression in fetal and adult gingival cells; Oda D et al.; Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium . The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue . Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues . K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells) . K1 and K10 were expressed in tissue, but not in cultured cells . The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5 . K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue . K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells . K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less . Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions . Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca2+ is shown by the expression of K13.

J Gen Virol, 1990 Jun, 71 ( Pt 6), 1391 - 4
Antigenic modulation of measles subacute sclerosing panencephalitis virus in a persistently infected rat glioma cell line by monoclonal anti-haemagglutinin antibodies; Zinnheimer-Dreikorn J et al.; Rat glioma C6 cells persistently infected with measles subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) express the viral membrane proteins haemagglutinin (HA) and F on their cell surface as well as the intracellular proteins N, P and M . Previously we have shown that the addition of a polyclonal antibody against the HA antigen to the growth medium of C6/SSPE cells led to a gradual loss of all viral antigens . Here we show that the addition of a monoclonal antibody (MAb K83) leads only to a transient decrease in viral antigens during the first three passages . After the third passage viral antigens start to increase and after five passages they produce more antigens than at the premodulation level . At this point of the MAb treatment, MAb K83 no longer recognized the HA antigen on the surface of the cells and in virus particles produced by these cells in contrast with polyclonal antibodies or other MAbs against the HA antigen . The results suggest that specific variants of the SSPE virus with an altered HA antigen were selected by the MAb treatment.

Biochim Biophys Acta, 1990 May 24, 1024(2), 307 - 17
Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium; Comiskey SJ et al.; A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively . The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid . In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes . The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum . Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes . In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability . Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures . The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media . The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay . Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium . However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.

Arch Biochem Biophys, 1990 May 15, 279(1), 116 - 21
Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA; Huskey SE et al.; An expression plasmid, pKK-GTB2, containing the complete coding sequence of a rat liver glutathione S-transferase Yc subunit was constructed and expressed in Escherichia coli . The entire Yc cDNA sequence from plasmid pGTB42 (Telakowski-Hopskins et al., 1985, J . Biol . Chem . 260, 5820-5825) was amplified by the polymerase chain reaction, subcloned into modified expression vector A6316 (Schoner et al., 1986, Proc . Natl . Acad . Sci . USA 83, 8506-8510 and Linemeyer et al., 1987, Bio/Technology 5, 960-965) and transformed into E . coli strain AB1899 . The colonies were screened by hybridization to pGTB42 and the production of Yc subunit was detected by immunoblot analysis . The purified recombinant Yc subunit was active in the conjugation and peroxidation reactions, and appeared homogeneous as judged by sodium dodecyl sulfate gel electrophoresis . Amino acid sequencing of the expressed Yc subunit revealed that about 40% of the expressed protein was blocked at the N-terminus . Approximately 25% of the sequenceable protein (15% of total protein) contained the initiation methionine residue at the amino terminus whereas the rest of the sequenceable protein had proline as the N-terminus . In contrast, only one molecular species with Pro as the first amino acid was identified when the inducer isopropyl-beta-D-thiogalactopyranoside was omitted in the growth medium . Our observation indicated that under certain growth conditions, the enzymes responsible for protein maturation were not able to complete the processing of the overproduced recombinant Yc in E . coli.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 867 - 74
The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis; Bayer ME et al.; The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser . Under standard growth conditions and neutral pH all cells displayed a negative EPM . The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM . Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67 . No difference in the EPM was observed between rapidly growing and stationary-phase E . coli B . De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E . coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media . Extrusion of filamentous bacteriophage f1 from cells of its host, E . coli A95, caused a shift to a higher negative EPM . We also measured a variety of Gram-positive strains, all of which displayed different EPMs . When membrane fractions of E . coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed . The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.

FEMS Microbiol Lett, 1990 May, 57(1-2), 61 - 6
A method for the study of fungal growth inhibition by plant proteins; Ludwig A et al.; A bioassay is described for the study of inhibitory activity of plant proteins on fungal growth . Fungal spores were germinated in liquid growth medium and pipetted into wells of a microtitre plate . Fungal growth was followed spectrophotometrically . The bioassay was tested using crude protein extracts from plant tissues known to have high activities of chitinase and beta-1,3-glucanase, and with purified enzymes . Crude protein preparations and combinations of the purified enzymes produced a temporary reduction of growth but no permanent growth inhibition.

Int J Parasitol, 1990 May, 20(3), 299 - 305
Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro; Eisemann CH et al.; The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro . One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival . Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media . No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments . ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant . The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media . These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.

Appl Environ Microbiol, 1990 May, 56(5), 1221 - 8
Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp . succinogenes S85; Huang L et al.; Fibrobacter succinogenes subsp . succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture . During growth on cellulose, there was no accumulation of soluble carbohydrate . When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion . Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis . Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate . Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions . These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.

J Cell Physiol, 1990 May, 143(2), 381 - 90
Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane; Magnusson KE et al.; We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP) . Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period . Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal . In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D {x 10(8)} = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D {x 10(9)} = 2 cm2s-1; R = 60-70%) . However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells . By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells . It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.

J Neurochem, 1990 May, 54(5), 1666 - 76
Production of 1,2-diacylglycerol in PC12 cells by nerve growth factor and basic fibroblast growth factor; Altin JG et al.; The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with {3H}inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min . The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3) . Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3 . The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium {Dulbecco's modified Eagle's medium (DME) plus serum}, it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol % . The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min . This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM) . Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF . Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.

Indian J Exp Biol, 1990 May, 28(5), 430 - 3
Effect of pH stress on lipid composition of Saccharomyces cerevisiae; Singh B et al.; Total lipids (% dry weight basis) of S . cerevisiae increased when pH of the growth medium was altered . Phospholipid content increased when the yeast was grown at a pH higher than its optimal (pH 6) . Sterol content was not affected much . Sterol:phospholipid ratio was not affected by pH of the medium . Phosphatidylcholine content of S . cerevisiae was inversely related to pH of its growth medium . Glycolipids were more when the yeast was grown at pH 9 . Fatty acids of S . cerevisiae grown at pH 3 were more saturated which makes the membranes less fluid.

Mol Cell Biol, 1990 May, 10(5), 2294 - 301
Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae; Dancis A et al.; The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells . In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation . We report here the isolation of a mutant of S . cerevisiae lacking the reductase activity . This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation . Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1 . Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S . cerevisiae . This fragment contains FRE1, the wild-type allele of the mutant gene . The level of the gene transcript is regulated by iron in the same was as the reductase activity . The ferrous ion product of the reductase must traverse the plasma membrane . A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells . Thus, iron uptake in S . cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

J Cell Sci, 1990 May, 96 ( Pt 1), 131 - 41
Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei; Douglas GC et al.; The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei . During the first 24 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies . This was accompanied by progressive changes in the pattern of desmoplakin staining . In single cells, desmoplakin was dispersed throughout the cytoplasm . As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation . A pavement-like staining pattern was maintained even at 5 days . Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated . When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally . However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation . While cells grown in Ham's/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium . Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration . While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15- to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release . These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.

J Bacteriol, 1990 May, 172(5), 2710 - 5
Transcriptional regulation of the heat shock regulatory gene rpoH in Escherichia coli: involvement of a novel catabolite-sensitive promoter; Nagai H et al.; A catabolite-sensitive promoter was found to be involved in transcription of the heat shock regulatory gene rpoH encoding the sigma 32 protein . Expression of lacZ from the operon fusion, rpoHp-lacZ, was partially inhibited by glucose added to the broth medium . Dissection of the rpoH promoter region allowed us to localize the glucose-sensitive promoter to the 110-base-pair (bp) segment directly upstream of the rpoH coding region . Experiments on lacZ expression from the set of fusions in cya (adenylate cyclase) and crp (cyclic AMP {cAMP} receptor protein) mutants also supported the involvement of a catabolite-sensitive promoter . Analysis of rpoH mRNAs by S1 nuclease protection experiments led us to identify a novel promoter, designated P5, that is regulated by cAMP and the cAMP receptor protein . Studies of rpoH transcription in vitro demonstrated that RNA polymerase-sigma 70 can transcribe from the P5 promoter only in the presence of cAMP and its receptor protein . The 5' ends of P5 transcripts obtained in vivo and in vitro were found to be at 61 to 62 bp upstream of the initiation codon, and a putative binding sequence for the cAMP receptor protein was found at 38 to 39 bp further upstream . Transcription from the P5 promoter is increased by the addition of ethanol to the growth medium; however, the increase is greater in the presence of glucose than in its absence . These results add a new dimension to the transcriptional control of rpoH and to the regulation of the heat shock response in Escherichia coli.

J Invest Dermatol, 1990 May, 94(5), 657 - 61
A comparison of the effects of antitumor agents upon normal human epidermal keratinocytes and human squamous cell carcinoma; Firestone WM et al.; The pharmacologic responses of normal and malignant epidermal cells to chemotherapeutic agents were examined in a system which consists of a serum-free "defined" medium . The effects of methotrexate (MTX), fluorodeoxyuridine (FUDR), and hydroxyurea upon cell growth, DNA synthesis, thymidylate synthase activity, and dihydrofolate reductase (DHFR) activity were compared in normal human epidermal keratinocytes (NHEK), newborn epidermal cells (NEC), human squamous cell carcinoma (SCC-25), and a methotrexate-resistant human squamous cell carcinoma (SCC-15R) . Normal keratinocytes were found to be 10(3) to 10(4) times less sensitive to the effects of MTX and FUDR than the malignant cells with respect to growth and DNA synthesis . The differential sensitivity to MTX and FUDR was not due to differences in growth media, increased target enzyme activity, e.g., DHFR and thymidylate synthase, or impaired transport of these drugs . The results indicate that the mechanism for the increased sensitivity of the squamous cell carcinoma to MTX and FUDR must involve a process which is distal to the de novo synthesis of dTMP.

J Clin Immunol, 1990 May, 10(3), 175 - 81
Immunosuppressive activity in human embryo growth media is associated with successful pregnancy: effect of gonadotropin releasing hormone agonist (GnRHa) treatment of patients undergoing in vitro fertilization and embryo transfer (IVF-ET); Bose R et al.; The aim of the present study was to determine whether the secretion of embryo-associated immunosuppressor factor (EASF) by preimplantation embryo correlates with pregnancy outcome and whether this relationship is influenced by pretreatment of gonadotropin releasing hormone agonist (GnRHa) in patients undergoing in vitro fertilization and embryo transfer (IVF-ET) . EASF activity was measured using concanavalin A-induced human lymphocyte proliferation assay in 256 embryo growth media obtained from 61 patients undergoing IVF-ET . EASF activity was then correlated with GnRHa treatment and pregnancy outcome in these IVF patients . Results indicate that (i) the presence of immunosuppressive activity in human embryo growth media is associated with success of pregnancy in GnRHa nontreated patients and (ii) serum factors present in GnRHa-treated patients may affect the EASF secretion by preembryos in vitro.

Arch Environ Contam Toxicol, 1990 May-Jun, 19(3), 395 - 8
Effect of environmental toxicants on enzyme biosynthesis: a comparison of beta-galactosidase, alpha-glucosidase and tryptophanase; Dutton RJ et al.; Except for beta-galactosidase, little is known about the effect of environmental toxicants on enzyme induction . The information could be potentially useful for the development of low-cost and rapid ecotoxicity assays . The effect of toxicants on the de novo biosynthesis of three inducible enzymes, beta-galactosidase and tryptophanase in E . coli and alpha-glucosidase in B . subtilis was investigated . Biosynthesis of alpha-glucosidase was the most sensitive to environmental toxicants, particularly pentachlorophenol and sodium dodecyl sulfate . The sensitivity of B . subtilis to toxicants was further increased when Tween 80 was incorporated in the growth medium.

Food Addit Contam, 1990 May-Jun, 7(3), 357 - 68
Mutagenicity and cytotoxicity of N-nitrosothiazolidine-4-carboxylic acid; Lin IN et al.; N-Nitrosothiazolidine-4-carboxylic acid (NTCA) was prepared by treating L-thioproline with sodium nitrite at pH 2, 37 degrees C . The compound was characterized by mass spectrometry, infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy . The cytotoxic and mutagenic properties of NTCA were explored by exposing the human cell line HeLa S3 at 37 degrees C to various concentrations (10 microM-10 mM) of the compound for various periods of time (1-36 h) and by monitoring its effects on cell viability, cell growth, intracellular metabolic activities such as DNA, RNA, and protein synthesis and on DNA repair synthesis ('unscheduled' DNA synthesis) . NTCA did not affect the cells' viability at any concentration or incubation period but decreased cell growth at the limiting concentration of 10 mM in the growth medium . NTCA had no effect on RNA and protein synthesis, and, similarly, it had no effect on DNA synthesis at concentrations up to 3 mM . Curiously, the stimulation of DNA synthesis by NTCA was seen at 10 mM after 24 h of incubation . NTCA did not initiate 'unscheduled' DNA synthesis (DNA repair) . It is concluded that the compound displays very little cytotoxicity and no mutagenicity in the HeLa S3 test system; hence, its presence in humans and in the human food supply is likely to be of little importance as far as its oncogenic properties are concerned.

Mutat Res, 1990 May, 241(1), 49 - 66
Botran and bleomycin induce crossing-over, and bleomycin also increases aneuploidy in diploid strains of Aspergillus; Kafer E; Both bleomycin, an antineoplastic drug, and botran (2,6-dichloro-4-nitro-aniline), a fungicide, are known to inhibit growth and induce genetic segregation in diploid tester strains of Aspergillus nidulans when present in agar media . To identify primary effects, samples of induced apparent crossover types were analysed in detail . For both compounds, coincident and consecutive events of mitotic crossing-over were found to be very frequent and such events showed a random distribution among isolated colour sectors . In the case of botran, a thorough search for imbalanced precursor types was negative and recessive lethals were found only rarely . Such segregants are therefore unlikely the result of terminal deletions . For bleomycin, induction of reciprocal crossing-over was confirmed by treatments of germinating conidia . On plating to normal growth medium, crossover segregants showed up as coloured half- or quarter-colonies, including some "twin spots" . Whole coloured colonies were also frequent and these increased with dose levels which caused decreasing survival and increasing frequencies of abnormal colonies . Analysis of large fractions of such "abnormals" identified aneuploids in all cases . While botran, in plate tests, also increased haploid segregants and disomic precursors could be found, tests of germinating conidia "in liquid" were inconclusive, because botran is insoluble in water . Some increases of aneuploids were observed, but only when botran and the solvent DMSO both were present at increased levels.

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