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 Saccharomyce