Br J Cancer, 1982 Feb, 45(2), 256 - 64 Oxygen tensions in multicell spheroids of two cell lines; Mueller-Klieser WF et al.; O2 tensions (Po2) were measured with microelectrodes in multicellular spheroids from EMT6/Ro and V-79-171-B cells . The measurements were performed in spheroids kept in flowing growth medium that was equilibrated with 5% CO2 and air at a temperature of 37 degrees C and contained 5.5 mM glucose . The recorded Po2 profiles are characterized by a diffusion-depleted zone surrounding the spheroids and by a steep drop in Po2 within the spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids respectively . Smaller spheroid exhibit parabolic Po2 profiles, larger ones show a central plateau . The region of the steep decrease in Po2 corresponds to the thickness of the viable rim: the plateau region is created by the absence of O2 consumption in the central necrotic area . Po2 in the centre of EMT6/Ro spheroids decreased from 66 mmHg at a diameter of 400 micrometer to 13 mmHg at a diameter of 1000 micrometer . Under the present conditions during growth and in the experiments, values below 5 mmHg were recorded only in spheroids 1200 micrometer . Comparably low Po2 was recorded in V-79 spheroids with diameters of 650 micrometer + . In spheroids of this cell type with a diameter of 400 micrometer, Po2 was 42 mmHg . The findings provide evidence that necrosis may arise at average Po2 of 57 and 42 mmHg in EMT6/Ro and V-79-171B spheroids, respectively, grown under the conditions described. Cancer Treat Rep, 1982 Feb, 66(2), 327 - 38 Isolation and properties of Chinese hamster lung cells resistant to ellipticine derivatives; Salles B et al.; Chinese hamster lung cells resistant to 9-OH-ellipticine (9-OH-E) were selected in vitro by adding stepwise increasing drug concentrations to the cell growth medium . This selection procedure resulted in the isolation of two sublines, about tenfold and 12-fold resistant to 9-OH-E . This level of resistance did not increase even after about 30 months of drug exposure . Cytogenetic studies revealed that the resistant cells carry several discrete karyotype modifications, the most striking being the absence of one No . 5 chromosome and the presence of a marker chromosome characterized by peculiar G-banding . Development of 9-OH-E resistance was also associated with some changes in cell properties such as modifications of morphology and growth parameters, lower oncogenic potential, and cross-resistance to a variety of antitumor agents . Although these results suggest that the resistance is associated with a modification of cell membrane properties, drug uptake studies did not show any significant difference between the parental sensitive cells and the resistant sublines. Biokhimiia, 1982 Feb, 47(2), 284 - 9 {Changes in proteinase A and B activity during glucose repression in the yeast Saccharomyces cerevisiae}; Novikova LA et al.; The divisible and indivisible yeast S . cerevisiae were subjected to glucose repression by increasing the glucose content in the growth medium up to 10% . Under these conditions the total activities of both proteinases did not change significantly, while those of free (i.e . not bound to natural inhibitors) proteinases were increased manyfold . This effect is probably due to liberation of the proteinases from the vacuoles and digestion of cytosolic proteinase inhibitors. Biochem Genet, 1982 Feb, 20(1-2), 17 - 28 Biochemical evidence for diverse etiologies in biotin-responsive multiple carboxylase deficiency; Packman S et al.; Biotin-responsive multiple carboxylase deficiency can be categorized by clinical criteria into a neonatal-onset disorder and distinct syndrome of infantile onset . Pedigrees in each instance are consistent with autosomal recessive inheritance . For a neonatal-onset proband, the sensitivity to relative biotin deprivation and the rapid clinical response to biotin supplementation are reflected by in vitro studies . Specific activities of biotin-dependent pyruvate carboxylase, propionyl CoA carboxylase, and 1-methylcrotonyl CoA carboxylase are 0.8 to 16% of mean control values after growth of fibroblasts in intermediate and very low biotin concentrations . Following relative biotin depletion, pyruvate carboxylase activity returns to normal after only 14 hr of growth in biotin-supplemented medium . In contrast, carboxylase activities in fibroblasts of an infantile-onset proband remain normal at very low biotin concentrations, even when avidin is added to the growth medium . The clinical heterogeneity, taken together with the distinct responses of cultured skin fibroblasts to biotin deprivation in vitro, probably reflect fundamentally different etiologies for the two categories of biotin-responsive multiple carboxylase deficiency. J Bacteriol, 1982 Feb, 149(2), 469 - 78 Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli; del Campillo-Campbell A et al.; The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme . The products of four other E . coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity . Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively . The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium . Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold . This effect is reversed by the addition of 1 mM molybdate to the growth medium . The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes . The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage . Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor. Eur J Biochem, 1982 Feb, 122(1), 169 - 74 Phospholipid interconversions in Mycoplasma capricolum; Gross Z et al.; Mycoplasma capricolum cells increase their phospholipid content by incorporating exogenous phospholipids from the growth medium . Growing the cells in media with increasing serum concentrations resulted in a massive incorporation of phosphatidylcholine and sphingomyelin (up to about 50% of total phospholipids) into the cell membrane . The incorporation of the exogenous phospholipids had essentially no effect on the rate of cell growth and did not decrease the overall phospholipid biosynthesis of the cells . Thus, the ratio of phospholipid to protein in membranes from cells grown with 5% horse serum was 0.5 (mumol/mg) compared to 0.3 (mumol/mg) in cells grown without serum, and the relative content of charged polar lipids was apparently decreased . The consequence of the incorporation of exogenous phosphatidylcholine was an alteration in the relative amount of the major end-products of the de novo phospholipid biosynthesis; a marked increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol was observed . The possibility that the increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol is part of a control mechanism to maintain a mixture of bilayer and non-bilayer lipids is discussed. Biochim Biophys Acta, 1982 Jan 22, 684(2), 241 - 8 Cell surface of the fish pathogenic bacterium Aeromonas salmonicida . I . Relationship between autoagglutination and the presence of a major cell envelope protein; Evenberg D et al.; A comparison was made of membrane protein patterns of various Aeromonas salmonicida strains, initially isolated from different habitats with respect to fish species affected, pathological entity, and geographic location of the outbreak of the disease . A major protein with a molecular weight of 54 000 was found in all autoagglutinating strains, whereas this protein is present in low amounts, or not at all, in non-autoagglutinating strains . Evidence for a causal relationship between the presence of this protein and the phenomenon of autoagglutination came from the observation that a change of the growth medium led simultaneously to an almost complete loss of the additional cell envelope protein and the property of autoagglutination . As it has already been reported that autoagglutination is correlated with the presence of an additional cell surface layer, we hypothesize that the additional cell envelope protein is the (major) subunit of this layer . The application of the gel immuno radio assay, an immunological technique suited to detect antigens in a gel, revealed that the additional cell envelope proteins of all tested strains are immunologically related . The possibility to the use of this protein as a component of a vaccine against A . salmonicida infections is discussed. Biochim Biophys Acta, 1982 Jan 12, 714(1), 93 - 100 Regulation of putrescine metabolism in Ehrlich ascites carcinoma cells exposed to hypotonic medium; Kapyaho K et al.; When exposed to hypotonic growth medium, Ehrlich ascites carcinoma cells showed a rapid stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in 4 h, followed by a rise in their putrescine content . This effect was totally abolished by addition of a slightly hypertonic concentration of sodium chloride or sucrose to the medium . The general protein synthesis was unaffected by the hypotonic treatment . The uptake of putrescine and, to a lesser extent, spermidine was enhanced, the conversion of the radioactive putrescine into spermidine appeared partially inhibited during later stages of the hypotonic treatment . As a result, the half-life of putrescine increased from 2.8 h under isoosmotic conditions to 7.3 h in hypoosmotic medium . Both exogenous ({14C}-putrescine-derived) and endogenous ({14C}ornithine-derived) putrescine degraded at similar rates in control and hypotonic cells, yet the putrescine taken from the medium degraded preferably to nonpolyamine products, while the putrescine synthetized in the cell was converted evenly to spermidine and to other metabolites . Adenosyl-methionine decarboxylase activity (EC 4.1.1.50), which provides the second precursor for spermidine and spermine synthesis, was distinctly inhibited in the hypotonic medium . Inhibition was likewise observed in spermidine synthase activity, while spermine synthase was marginally stimulated . It appears that the hypotonic treatment serves a special condition under which not only the formation of putrescine is enhanced dramatically but the cells also attempt to converse the diamine by preventing its further metabolism to higher polyamines. Pharmacology, 1982, 25(3), 170 - 6 Response of mouse liver tumor cells to the differentiation-inducing agent dimethylsulfoxide; Higgins PJ; The effects of the differentiation-inducing agent dimethylsulfoxide (DMSO) on the growth and protein composition of murine hepatic tumor cell (BW77-1) cultures were investigated using various concentrations of the polar solvent in the growth medium . A 4-day exposure to DMSO in concentrations of 0.5, 1 and 3% reduced final hepatocyte population density to 63, 43 and 15% of control values, respectively, while the amount of transferrin in the cellular and extracellular compartments increased in a dose-dependent fashion . The biochemical basis for elevated transferrin extractable per 10(6) BW77-1 cells in DMSO-stimulated cultures was due to increases in both the transferrin contribution to total cellular protein and the mean protein content per cell . This stimulation in cellular protein levels was reflected in the incorporation of {3H}-amino acids into cell-associated 10% trichloroacetic acid-insoluble material . Cultures of transformed liver epithelial cells thus appear to respond to in vitro DMSO exposure by increasing their accumulation of a late-stage hepatocyte gene product. Z Allg Mikrobiol, 1982, 22(6), 379 - 88 {Cytochrome pattern of methylotropic acetic acid bacterium MB 58 as dependent on growth conditions}; Steudel A et al.; In contrast to methylotrophic bacteria investigated up to now the facultative methylotrophic Bacterium MB 58 (Acetobactersp . MB 58) does not possess a cytochrome aa3-complex, but we could find out cytochrome, cytochrome cco, cytochrome a1, and moreover cytochrome d in dependence on the growth conditions . Cytochrome d was found only in stationary phase of heterotrophic growth . Under methylotrophic growth conditions cytochrome d could be demonstrated only by lowering of the aeration rate during the fermentation, by variation the pH-value of the growth medium from 4.0 to 6.5 and with low growth rates (low dilution rates) during continuous fermentation . The addition of cyanide to the oxidized suspension of bacteria during the registration of the cytochrome-redox-difference spectrum allowed the selective representation of cytochrome d under all conditions even if no identification was possible in the spectrum normally . The oxidation of cytochrome d of Acetobacter sp . MB 58 in the presence of cyanide is an indication of its cyanide insensitivity . The low level of b-type cytochromes could be represented by a special technique for registration of spectra . In this connection a unknown absorption peak at 570 nm was registered . The cyanide insensitivity of cytochrome d from Acetobacter sp . MB 58 and the occurrence of several terminal oxidases is appreciated as a hint for a branched respiratory chain. Int Arch Allergy Appl Immunol, 1982, 69(3), 224 - 30 Isolation and characterization of the allergen Dpt 12 from Dermatophagoides pteronyssinus by chromatofocusing; Stewart GA; The allergen Dpt 12, from the house dust mite Dermatophagoides pteronyssinus, was isolated from spent growth medium by chromatofocusing . The allergen was isolated in the pI range 5.86-6.95, indicating heterogeneity with regard to pI . However, on sodium dodecylsulphate-polyacrylamide gel electrophoresis the allergen appeared homogeneous with an apparent molecular weight of 27,000 daltons . Radioallergosorbent scores utilizing paper discs coupled with Dpt 12 correlated significantly (p less than 0.0005) with those obtained using discs coupled with whole mite extract, although the corresponding titres were higher with Dpt 12 (p less than 0.0005). Mol Cell Biol, 1982 Jan, 2(1), 76 - 81 Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes; Gottlieb M et al.; An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes . The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture . The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates . The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000 . The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L . donovani promastigotes which has been previously described. J Cell Biochem, 1982, 18(1), 121 - 33 Differential reactivation of zinc-mediated metallothionein induction in ultraviolet-irradiated normal and repair-deficient human cells; Hildebrand CE et al.; The ubiquitous, low-molecular-weight, thiol-rich, metal-binding protein, metallothionein (MT), can be induced in cultured normal human fibroblasts (NF) and xeroderma pigmentosum (XP) cells by exposure to ZnCl2 . Both NF and XP cells tolerate up to 200 microM ZnCl2 in the growth medium, upon addition of ZnCl2 (200 microM) to monolayer cultures, both NF and XP cells showed similar kinetics for the induction of MT synthesis: Within 7 hours the MT synthesis rate rose from a low, marginally detectable rate to a maximal rate at least 50-fold greater than the basal rate . The induction of MT synthesis in both cell types was inhibited by actinomycin D (5 microgram/ml), indicating that the induction process is controlled at the level of transcription . Exposure of NF and XP cells to far ultraviolet light (UV) followed by induction with ZnCl2 resulted in a UV dose-dependent decrease in the he maximal rate of MT synthesis measured 8.5 hours postirradiation . The UV sensitivity of the MT induction was greater in XP cells than in NF cells . However, considerations of the differential repair capacities of NF and XP cells superimposed upon the kinetics of MT induction were invoked to explain the apparent differential UV sensitivity of MT induction . Liquid holding recovery experiments showed that NF cells possess the capacity to reactivate this inducible gene function rapidly while XP cells are deficient in the reactivation capacity . These results are discussed in the context of both UV transcriptional mapping of this inducible gene function and development of techniques for measuring repair of transcription-blocking lesions. J Bacteriol, 1982 Jan, 149(1), 338 - 45 Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis; Rottem S et al.; Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches . The patches appeared in cells quenched from either 4 or 37 degrees C . Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine . The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M . gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium . Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+ . Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M . gallisepticum membranes was almost identical to that of an aqueous dispersion of M . gallisepticum membrane lipids . Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions . The possibility that the osmotic resistance of M . gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed. J Bacteriol, 1982 Jan, 149(1), 131 - 5 Iron transport in Mycobacterium smegmatis: Uptake of iron from ferric citrate; Messenger AJ et al.; In mycobacterial growth medium 40 to 400 microM citrate was required to solubilize 2 microM 55Fe . This solubilized 55Fe was taken up into both iron-deficient and iron sufficient washed cell suspensions of Mycobacterium smegmatis and Mycobacterium bovis BCG . Although the 55Fe was taken up into the cell, the citrate was not . The uptake system with M . smegmatis was not inhibited by electron transport inhibitors, uncouplers of oxidative phosphorylation, or thiol reagents and was saturable with iron at approximately 35 microM . The system was independent of the iron transport systems already known to exist in M . smegmatis: i.e., the two exochelin routes of assimilation as well as the mycobactin-salicylate system . It was not induced by the presence of 400 microM citrate in the growth medium, nor did the presence of citrate in the medium affect the production of either exochelin or mycobactin. Mol Gen Genet, 1982, 186(2), 157 - 63 Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase; Greenberg ML et al.; The enzyme inositol-1-phosphate synthase is repressed at least 50-fold in wild type yeast grown in inositol-supplemented media . Mutants which synthesize this enzyme constitutively have been isolated using a selection procedure based on excretion of inositol into the growth medium by putative mutants . Biochemical analysis of one of the mutants (opi1-1) confirmed that the nature of the mutations is regulatory, and not in the structural gene for the enzyme . Immunoprecipitation of crude extracts with antibody directed against purified inositol-1-phosphate synthase showed that a protein which reacts with the antibody is present in the mutant grown under both repressing and derepressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed . Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and derepressing conditions . Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD . These analyses indicated that purified mutant synthase was similar to the wild type enzyme. Comp Biochem Physiol B, 1982, 71(3), 397 - 402 Fatty acids of Trypanosoma cruzi; Timm SL et al.; 1 . The fatty acid pattern of total lipids from T . cruzi is different from one of its growth medium . 2 . The distribution of major fatty acids in phospholipid fraction was linoleic (50.4%), oleic (25.6%), stearic (10.1%) and palmitic (6.3%) and in neutral lipid fraction oleic and linolelc (about 29% each), palmitic (18.3%) and stearic (9.8%) . 3 . In each of the individual phospholipids a particular fatty acid pattern was observed . 4 . For phosphatidylcholine: linoleic (55.7%), oleic (16.6%), stearic and palmitic (about 8% each) and polyunsaturated acids (4%) . 5 . For phosphatidylethanolamine: linoleic (41.5%), oleic (27.6%), stearic (9.9%) and an unidentified fatty acid (9.2%) . 6 . For phosphatidylinositol: stearic and linoleic (about 30% each) and oleic (22.1%). Biosci Rep, 1982 Jan, 2(1), 15 - 30 Calcium ions and the control of proliferation in normal and cancer cells; Durham AC et al.; Several lines of evidence suggest tha Ca2+ ions control cell proliferation: Ca2+ entry into cytoplasm acts as a general mitogen; serum and serum-replacements induce Ca2+ influx; the Ca2+ concentrations in growth media required to support the proliferation of normal cells are much higher than those required for cancer cells; serum and growth factors reduce the Ca2+ requirements of normal cells; tumour promoters alter Ca2+ fluxes via a mechanism used principally by growth factors . Minor supporting evidence includes the effects of various drugs and viruses, and the behaviour of tumour cell mitochondria and intercellular junctions . It is still not possible to decide exactly where and when inside cells the critical effect of Ca2+ on proliferation occurs, but we discuss at length the practical problems of understanding Ca2+ movements in tissue-culture cells . Carried to its logical conclusion, present evidence suggests that an overridden or bypassed Ca2+ control process may be the key, common determinant of unrestrained proliferation in cancer cells. J Cell Biochem, 1982, 20(1), 41 - 50 Involvement of multiple subcellular compartments in intracellular processing of epidermal growth factor; Miskimins WK et al.; The intracellular translocation and processing of epidermal growth factor (EGF) in 3T3 cells has been studied utilizing Percoll density gradients . EGF is internalized and rapidly becomes associated with two types of intracellular compartments . The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition . In growth medium, an increased proportion of EGF is taken up into a Golgi-like element . Uptake through this pathway correlates with a decrease in degradation of the ligand . In the absence of serum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05 . Uptake through this pathway correlates with increased degradation of the ligand . The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes . In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation . In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound . The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor. Arch Virol, 1982, 74(1), 11 - 20 The effect of cytochalasin D and monensin on enveloped vaccinia virus release; Payne LG et al.; Both monensin and cytochalasin D reduced the production of infectious cell associated virus and infectious extracellular virus with the latter clearly being the more sensitive . The difference in yields were even more clearly seen if the yield of virus particles was monitored instead of yield of infectious virus . Addition of 1 microgram/ml cytochalasin D or 1 microM monensin to the growth medium of vaccinia virus-infected cells inhibited the appearance of extracellular enveloped vaccinia virus (EEV) in the growth medium without affecting the production of intracellular naked vaccinia virus (INV) particles . Although EEV was not released into the medium of cytochalasin D treated cells, EEV was, nevertheless, detectable in CsCl gradients of cell associated virus . Monensin treatment did not affect the synthesis of vaccinia glycoproteins but did significantly reduce the transport of these glycoproteins to the cell surface and also reduced the secretion of proteins . Monensin had the additional effect of causing the appearance of numerous large vacuoles in the cytoplasm . Vaccinia is normally wrapped by cytoplasmic membranes in preparation for release . The monensin induced conversion of cytoplasmic membranes to large vacuoles is presumably the basis for the block in virus wrapping and subsequent release . Cytochalasin D did not alter any of the steps in protein synthesis, transport or secretion . Electronmicroscopic studies confirmed the existence of EEV on the surface of infected cells treated with cytochalasin D . This drug which specifically affects cellular microfilament organisation thus imposes a block on the final release of EEV from the cell surface. J Cell Physiol, 1982 Jan, 110(1), 9 - 16 Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells; Ager A et al.; Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium . Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium . Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar . In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2 . Experiments with {14C} prostaglandin H2 or {14C}arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2 . Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density . The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production . These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment. J Bacteriol, 1982 Jan, 149(1), 361 - 3 Accumulation of poly-beta-hydroxybutyrate in Spirulina platensis; Campbell J 3rd et al.; Poly-beta-hydroxybutyrate has been identified in the cyanobacterium Spirulina platensis . The addition of reduced carbon compounds to the growth medium was not required for poly-beta-hydroxybutyrate accumulation . Poly-beta-hydroxybutyrate accumulated during exponential growth to 6% of the total dry weight and then decreased during the stationary phase. Microbios, 1982, 33(133-34), 149 - 60 Growth studies on Mycobacterium BCG: establishment of growth curves and measurement of the oxygen tension of the growth medium; Moore DF et al.; Mycobacterium BCG grew exponentially in shallow, static volumes of culture medium for approximately 10 days; the oxygen tension of the medium at all stages of growth was 100% saturation . Higher yields were obtained from Dubos than from glycerol-free medium . In static cultures, the oxygen tension of the culture and consequently the growth rate of BCG was dependent on the depth of the medium; active growth ceased at an oxygen tension of less than 40% saturation . BCG grew actively in a cell sediment, while cells growing in suspension made a negligible contribution to the yield. Genetika, 1982, 18(8), 1245 - 54 {Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation . II . Study of DNA damage and repair in gamma-irradiated cells}; Bresler SE et al.; Formation and repair of single-stranded breaks (ssb) and double-stranded breaks (dsb) in DNA of gamma-irradiated cells of three Escherichia coli strains (the parental strain AB1157 and radiation-resistant mutants Gamr444 and Gamr445) were studied by centrifugation in alkaline or neutral sucrose gradients . The initial yield of ssb and the kinetics of their repair during post-irradiation incubation in a growth medium are similar for Gamr mutants and the wild-type strain . The yield of dsb in the chromosome of Gamr mutants is significantly lower than in chromosomal DNA of the parental strain, both immediately after gamma-irradiation and after three hours of post-irradiation incubation in a growth medium . The decreased yield of dsb in the Gamr mutants correlates with their relative radioresistance . It was found also that the level of DNA degradation is significantly lower in UV- or gamma-irradiated cells of Gamr mutants, as compared with the wild-type strain . It is suggested that enhanced radioresistance of the Gamr mutants is due to decreased formation of enzymatically induced dsb, as a consequence of moderated DNA degradation under the action of repair exonucleases and also to enhanced efficiency of dsb repair. EMBO J, 1982, 1(7), 801 - 4 An ion-channel forming protein produced by Entamoeba histolytica; Lynch EC et al.; We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery . A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction . Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes . Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude . The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV . In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease . Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g . It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS . Under these conditions it has an apparent mol . wt . of 13 000 daltons. EMBO J, 1982, 1(3), 333 - 7 Regulation of adenylate cyclase in E . coli; Gstrein-Reider E et al.; The intracellular concentrations of cAMP in Escherichia coli are regulated mainly by control of the activity of adenylate cyclase . Withdrawal of the carbon source from the growth medium causes a gradual reduction of cellular energy and a dramatic stimulation of cyclase activity . Manipulations of the proton gradient at the cell membrane of ATP synthase-deficient E . coli (unc-) revealed that this part of the energy compartment is not responsible for the starvation-induced stimulation of cyclase . Neither is the ATP pool involved in regulation of the activity of the cyclase . The intracellular concentrations of ATP were experimentally lowered by purine starvation of auxotrophs, by inhibition of purine synthesis using amethopterin, or by affecting ATP synthesis using arsenate . None of these conditions led to stimulation of cyclase activity . The control of cyclase is exerted not via the energy pools but via uptake systems of energy substrates independent of whether the substrate can be metabolized or not, or how the transport is energized . The stringent coupling between these transport systems and cyclase activity enables the cell to react instantaneously to changes in its environment. Folia Biol (Praha), 1982, 28(5), 311 - 6 Inhibitory activity of cytosine arabinoside on Marek's disease virus; Benda V; The presence of ara-C in growth medium at concentrations of 10(-4) to 10(-7) M completely or partially suppressed the formation of plaques specific for MDV or HVT and decreased proportionally the growth of HPRS line 1 lymphoblastoid cells . Administration of ara-C to chickens immediately after infection with MDV (1 mg/chicken/day i.p . for 5 days) reduced the incidence of Marek's disease by 50% . Thus ara-C appears to be an inhibitor of Marek's disease. Mol Gen Genet, 1982, 186(4), 482 - 7 Negative control of ornithine decarboxylase and arginine decarboxylase by adenosine-3':5'-cyclic monophosphate in Escherichia coli; Wright JM et al.; The polyamine biosynthetic enzymes, ornithine decarboxylase (EC 4.1.1.17) (ODC) and arginine decarboxylase (EC 4.1.1.19) (ADC), are negatively controlled by cAMP in Escherichia coli . The specific activities of ODC and ADC were determined in crude extracts prepared from E . coli strains carrying a mutation in the adenylate cyclase (EC 4.6.1.1) structural gene (cya) and wildtype strains . These strains were cultured on various carbon sources in the presence and absence of cAMP . In wild-type strains, ODC and ADC activities were diminished in cells grown on glycerol compared to these strains cultured on glucose . When cya strains were grown on glucose or glycerol, ODC and ADC activities were the same . Addition of 1 mM cAMP to glucose-based medium repressed ODC and ADC activities in both the wild-type and cya strains . Furthermore, cAMP exerts its negative control through the cAMP receptor protein, since strains carrying a mutation in the crp structural gene fail to repress ODC and ADC activities in response to increased cAMP obtained by carbon source manipulation or cAMP supplementation of the growth medium . This evidence suggests that negative control of ODC and ADC by cAMP occurs at the level of transcription. J Cancer Res Clin Oncol, 1982, 103(1), 17 - 29 Induction of differentiation in human promyelocytic leukemia cells by tumor promoters; Nakayasu M et al.; 12-)-Tetradecanoylphorbol-13-acetate (TPA), the prototype polyfunctional diterpene ester tumor promoter of two-step carcinogenesis in mouse skin, induced differentiation of human promyelocytic leukemia cells (HL-60) in culture . Differentiation of HL-60 cells was characterized by increased phagocytosis, increased lysozyme activity (EC 3.2.1.17) in the growth medium, and changes in morphology to those characteristics of more mature cells resembling macrophages . Many of the cells treated with TPA became aggregated, attaching firmly to culture flasks . The average intracellular myeloperoxidase activity (EC 1.11.1.7) per cell decreased during induction of differentiation by TPA . It was also found that TPA enhanced, rather than inhibited, differentiation of HL-60 cells induced by DMSO . In addition to TPA, several polyfunctional diterpene esters of the tigliane, ingenane, and daphnane type have been tested for their ability to induce morphological and functional changes of HL-60 cells . The activities of the compounds to induce these changes correlated well with their activities as tumor promoters in two-step carcinogenesis in mouse skin . In particular, half the concentrations required for induction of adhesion of the cells to flasks were roughly correlated to the potency of these compounds as tumor promoters . Among the compounds tested, phorbol-12,13-didecanoate (PDD), ingenol-3-hexadecanoate, Pimelea factor P1 and Pimelea factor P2 were as active as TPA, while 4-O-methyl-TPA and 4 alpha-PDD were much less active . Phorbol and ingenol were totally inactive up to a concentrations 10,000-fold higher than that of TPA. Vopr Virusol, 1982 Jan-Feb, (1), 34 - 8 {Hormone-dependent process of the maturation of the major structural proteins of the mouse mammary tumor virus}; Beriashvili MM et al.; The following findings were obtained by the radio-immunoprecipitation method with antisera to gp52 and p27 . When the cells were cultivated in a hormone-free medium, they contained precursors of proteins of gene gag (Pr 73gag) and gene evn (gPr 70env) . No mature structural MTV proteins were found . The addition of insulin to the growth medium had no effect on maturation of protein precursors . When dexamethazone was added to the medium, gPr 70env "maturated" to gp52 of the main envelope glycoprotein of virion coat whereas Pr 73gag was not cleaved into final products . When the cells were cultivated in the presence of insulin and dexamethazone, there was a marked stimulation of Pr 73gag (although its maturation did not occur), g Pr 70env, and, especially, gp 52 . Extracellular particles were found to contain p27 which was precipitated by homologous monospecific antiserum, that is, in the system of clone F2 cells processing of Pr 73gag occurred mainly in extracellular particles . Thus, expression of all three known genes (gag, pol, and env) occurred in the cells of cloned F2 culture. Folia Microbiol (Praha), 1982, 27(2), 76 - 80 Relationship between the composition of phospholipids and respiratory activity of choline-deficient mutants of Neurospora crassa; Fecikova H et al.; Phosphatidylcholine is one of the most frequent phospholipid components of the inner mitochondrial membrane of Neurospora crassa . Quantitative analysis of phosphlipids of the wild strain of Neurospora crassa and of its two cho mutants showed that these strains did not significantly differ in the content of phosphatidylcholine . Mutants cultivated in a medium without choline contained, as compared with the wild strain, an increased amount of phosphatidylserine and a decreased quantity of phosphatidic acid . Respiratory activity increased and sensitivity to inhibitors of respiration changed . It is likely that the presence of choline in the growth medium exerts a regulatory effect on the cell metabolism of these mutants. Arch Dermatol Res, 1982, 273(1-2), 9 - 14 Pemphigus antibodies fixation and keratinocyte differentiation in organ cultures . Effects of some agents influencing the intracellular content of cyclic AMP; Sbano E et al.; The effect of some agents, influencing the cyclic adenosine 3', 5'-monophosphate (cAMP) content of human cells, on the ability of the keratinocytes of binding pemphigus antibodies was studied by using tissue cultures of rabbit esophagus . As demonstrated by immunofluorescence (IF) for IgG, the bound antibodies appeared markedly decreased on esophagus explants grown under standard conditions, that is without test agents, when compared to ones fixed on fresh esophagus . But the IF reaction was remarkably more intense when methylxanthines or epinephrine were added to the growth medium of the cultures . Following the addition of these agents to the cultures some histologic modifications appeared in the explants, indicating that the keratinization process had probably been stimulated . This temporal relationship of immunofluorescence and histologic findings seems to suggest the hypothesis that keratinocyte differentiation, regulation of cAMP intracellular content, and pemphigus antibodies fixation are related processes. Histochemistry, 1982, 76(1), 113 - 21 Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method; Severin E et al.; Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry . The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine . The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated . The chromosomal fluorescence intensity is reduced one step per generation . The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure . This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence . Therefore, the entire DNA content of these chromosomes now becomes measurable . The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift . In this way we could establish 3 successive mitoses . Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle. Arch Virol, 1982, 71(4), 311 - 22 Characterization of a hamster brain cell line persistently infected with measles virus; Vainionpaa R et al.; A persistent infection of measles virus in hamster brain cell cultures was established . Hamster brain cells were transformed with a human papovavirus strain BK and infected with a wild-type measles virus in order to get the cell line persistently infected with measles virus . About 75 per cent of these M-HB/MVB-cells were measles virus-infected . The cells released only small amount of infectious virus and produced low levels of interferon-like activity into the growth medium . During the first 50 passages no large syncytia typical of a lytic measles virus infection were seen . The products of measles virus infection in the cells and in cell culture fluids were followed at two temperatures . Another cell line persistently infected with measles virus (Lu-carrier-cells, 28) was investigated in parallel . In both cell lines measles antigens were cytoplasmic, but during the observation period large amounts of measles nucleocapsid accumulated in the nuclei of the M-HB/MVB-cells . The virus-specific protein synthesis in M-HB/MVB-cells was weak and the intracellular amount of immunoreactive measles nucleocapsid was only 50 per cent (600 ng/10(5) infected cells) of the (1200 ng/10(5) cells) found in Lu-carrier-cells . Also the release of nucleocapsid was minimal in hamster brain cells . The decreased temperature had no clear-cut effect on virus-specific protein synthesis or on the release of the virus-specific products. J Natl Cancer Inst, 1982 Jan, 68(1), 27 - 33 In vitro reversal of depressed T-lymphocyte function in the peripheral blood of brain tumor patients; Wood GW et al.; Peripheral blood mononuclear cells from some brain tumor patients exhibited depressed T-lymphocyte responses to the polyclonal mitogen phytohemagglutinin (PHA) . These responses, initially depressed, were restored to near normal by a 24-hour preculture in growth medium . The effect of preculture was mimicked by mild trypsinization before addition of PHA . In addition, cells treated with deaggregated antihuman IgG resulted in greatly reduced responses to mitogen by mononuclear cells from all brain tumor patients . Anti-IgG had no effect if cells were precultured for 24 hours . The results suggest that, in brain tumor patients, depressed immune function associated with tumor progression was caused by suppressor cells which were activated by humoral factors that express IgG antigenic determinants. J Cell Biochem, 1982, 20(4), 369 - 80 Influence of growth conditions on the composition of the plasma membrane from yeast and on kinetic properties of two membrane functions; Stadtlander K et al.; The influence of different growth conditions on the phospholipid composition and on two membrane functions, the Mg-ATPase and the purine transport system, was investigated . Addition of cholinechloride to the growth medium led to a certain rise in the amount of phosphatidylcholine, whereas supplementation with ethanolamine resulted in a considerably higher portion of phosphatidylethanolamine . When yeast cells were cultured at lower temperatures we found more short-chain fatty acids with a higher content of monounsaturated chains as compared to higher growth temperatures . Addition of paraquat, a herbicide which enhances lipid peroxidation by free radicals, reduced the amount of unsaturated fatty acids without influencing their chain length . The altered membrane composition had no influence on the basic mechanism of interaction between ATPase, MgATP, and free Mg2+ ions . However, several kinetic constants such as Km, Vmax, Ka, and especially Ki were influenced to some extent . Whereas the affinity of the purine transport system to its substrate was not significantly changed by the growth conditions, an effect on Vmax could be seen . Lower growth temperatures clearly led to higher maximal uptake velocities . The presence of paraquat during growth resulted in a considerable decrease of Vmax. Mol Gen Genet, 1982, 186(1), 33 - 9 Regulation of L-amino acid oxidase and of D-amino acid oxidase in Neurospora crassa; Sikora L et al.; Neurospora crassa possesses an inducible L-amino acid oxidase that is expressed only when cells are derepressed for nitrogen in the presence of an amino acid . Enzyme synthesis requires both induction by an amino acid and simultaneous nitrogen catabolite derepression . Carbon limition in the presence of an amino acid does not permit induction of L-amino acid oxidase . The nit-2 gene is a major regulatory locus which is believed to mediate nitrogen catabolite repression in Neurospora . Mutants of nit-2 are repressed for L-amino acid oxidase activity under conditions which lead to good enzyme induction in wild type and nit-2 revertants . The loss of the enzyme in nit-2 mutants does not result from inducer exclusion, which suggests that the nit-2 gene product has a direct role in controlling the expression of this enzyme . Substantial amounts of L-amino acid oxidase were detected in the growth medium as well as in cell extracts of the wild type strain . Biochemical data indicates that the intracellular and the extracellular L-amino acid oxidases are identical . Inhibitors of protein and of RNA synthesis block accumulation of L-amino acid oxidase, suggesting that enzyme expression is controlled at the level of transcription . D-amino acid oxidase can be detected in cell extracts of Neurospora grown in the presence of a D-amino acid . The enzyme is present in cys-3 mutants and is not repressed by high concentrations of sulfate or nitrogen indicating that D-amino acid oxidase is not a member of the sulfur or nitrogen regulatory circuits of this organism. Rev Esp Oncol, 1982, 29(4), 641 - 7 {Use of purine rescue pathways by L1210 cells exposed to methotrexate in vitro}; Buesa JM; Methotrexate (MTX) blocks the de novo synthesis of purines and pyrimidines due to a diminution of reduced folates, and produces an accumulation of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) . Phosphoribosyltransferases, in the presence of PRPP, synthesize nucleotides starting on purine bases, that are activated to triphosphates ("rescue pathway") . The authors study the activity of the rescue pathways in L1210 leukemia cells in vitro to restore the levels of ATP and GTP that have been lowered by MTX . Hypoxanthine (Hpx) was used as a preformed source of purines, high efficiency liquid chromatography was employed to estimate the intracellular concentrations of ATP and GTP . Two hours after 0.1 mM Hpx addition to in vitro L1210 cells in the presence of 0.1 microM MTX, the amount of ATP and GTP increased 5 to 7 times . In former experiments the author has shown that the uptake of PRPP increases when Hpx is added to L1210 cells exposed to MTX . The results of this work show that L1210 leukemia cells in vitro can use the rescue pathways of purine synthesis when Hpx is present in the growth medium, so preventing the anti-purine effect of MTX. J Biol Chem, 1981 Dec 10, 256(23), 12156 - 63 Ornithine decarboxylase from Saccharomyces cerevisiae . Purification, properties, and regulation of activity; Tyagi AK et al.; Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase . The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity . Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h . However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts . This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells. Lab Anim Sci, 1981 Dec, 31(6), 723 - 5 A technique for collection and cultivation of macrophages from Cynomolgus monkeys (Macaca fascicularis); Heisey GB et al.; Several techniques were compared for collection of peritoneal mononuclear phagocytes from cynomolgus monkeys . A multiple-holed cannula proved superior to a hypodermic needle for the collection of peritoneal washings because it was not occluded by the omentum . With the cannula technique 55--70% of the harvest medium could be recovered from the peritoneal cavity with a yield of 2.5--5 x 10(5) macrophages per ml . A closed system for injecting and collecting harvest medium was better than a syringe in preventing bacterial contamination of peritoneal washings . After collection, cells were resuspended in growth medium to a concentration of 5 x 10(5) macrophages per ml, distributed into Leighton tubes, and incubated at 36 degrees C . After 12 hours, cultures were washed to remove non-adherent cells and refed with growth medium . This technique provided a sterile, reliable method for the collection and cultivation of macrophages from monkeys. Can J Microbiol, 1981 Dec, 27(12), 1298 - 305 A defined growth medium for the production of chloroperoxidase by Caldariomyces fumago; Pickard MA; Ten strains of Caldariomyces fumago and related fungi were found to produce extracellular chloroperoxidase when grown on a glucose - malt extract medium . High enzyme levels and pigment production were observed for C . fumago ATCC 16373 and C . fumago CMI 89362 . Removal of malt extract from the medium and the replacement of glucose by fructose as the carbon source provided a defined medium which, by comparison with the complex medium, produced the following results with both fungal strains . Chloroperoxidase was produced to similar levels, with maximum production after 6 days rather than 12 days of growth; pigmentation of the medium was reduced by 90% and the pH of the medium remained constant, thus stabilizing enzyme activity . Addition or urea or proline as a nitrogen supplement to nitrate enhanced enzyme production by strain CMI 89362 . Comparison of the two strains indicated that CMI 89352 produced higher levels of chloroperoxidase than ATCC 16373. Eur J Cell Biol, 1981 Dec, 26(1), 154 - 7 Cytochalasin B inhibits polyamine biosynthesis in HeLa cells; Sunkara PS et al.; Cytochalasin B (CB), a potent inhibitor of cytokinesis in mammalian cells, has been shown to cause the rapid inhibition of ornithine decarboxylase and S-adenosyl methionine decarboxylase activities followed by a gradual but marked decline in intracellular levels of both putrescine and spermidine . Further, the multinucleation induced by CB could be reversed to some extent by exogenous addition of polyamines to the growth medium . The results of this study suggested that polyamines might have some indirect role in the CB-induced multinucleation of mammalian cells. Vet Res Commun, 1981 Dec, 5(2), 183 - 5 Cell lines from sheep and cattle blood; Woldehiwet Z et al.; A simple and reproducible method of establishing cell lines from the blood of sheep and cattle is described . Buffy coat cells were allowed to adhere to plastic culture flasks in media containing 20 per cent autologous plasma overnight . The fluids were then replaced with growth medium supplemented with non-inactivated foetal calf serum, lamb serum or autologous serum . Ovine cell lines were established with any of the serum supplements but bovine cell lines established more readily if unheated autologous serum was used. J Clin Microbiol, 1981 Dec, 14(6), 623 - 7 Comparison of liquid growth media for Legionella pneumophila; Saito A et al.; Ten liquid media were compared under standard conditions for their ability to support the growth of Legionella pneumophila . Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract yielded the best overall growth of the one strain of L . pneumophila examined . Growth rates were independent of pH changes which occurred during incubation . The growth rates of 10 different strains of L.pneumophila were compared in this medium . There appeared to be little difference in the growth rates of strains passaged frequently or infrequently, or between environmental and clinical isolates . Moderate aeration resulted in a faster growth rate and in approximately a 1 log10 higher final cell concentration as compared to a static broth culture . These experiments demonstrate that there are moderate to marked differences among the various media described in the literature and that no liquid medium yet developed supports rapid growth of L . pneumophila incubated without shaking. J Bacteriol, 1981 Dec, 148(3), 762 - 8 Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli; Takagi M et al.; Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide . Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E . coli K-10 . Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) . No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity . The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added . The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine {diS-C3-(5)}, indicating the generation of membrane potential . These results indicate that the reduction of trimethylamine N-oxide in E . coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force . Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants . Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it . Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants . Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system. Mol Cell Biol, 1981 Dec, 1(12), 1150 - 62 Membrane protein redistribution during differentiation of cultured human erythroleukemic cells; Hunt RC et al.; Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium . In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin . However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation . Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes . A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell . With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin. Acta Pathol Microbiol Scand {C}, 1981 Dec, 89(6), 353 - 8 The effect of uraemic serum and plasma on proliferating cells in vitro; Wessel-Aas T; Phytohaemagglutinin (PHA)-stimulated lymphocytes from healthy individuals and a malignant cell-line (K-562 cells) were cultured in a growth medium of 75 per cent RPMI 1640 (Gibco) supplemented with 25 per cent of uraemic serum or plasma obtained after systemic heparinization . Pooled human A Rh+ serum from 3 different donors served as controls . There was a depression of the DNA synthesis in both cell systems measured as uptake of methyl-3H-thymidine when the cells were cultured in uraemic milieu . Systemic heparinization markedly increased the depressive effect of uraemic plasma compared with controls . Heparin added to the growth media in concentrations much higher than those usually present in plasma after systemic heparinization had little effect on the cell proliferation . Systemic heparinization thus appears to induce production of toxic substances in plasma acting on proliferating cells. Infect Immun, 1981 Dec, 34(3), 888 - 94 Protection against feline leukemia by vaccination with a subunit vaccine; Lewis MG et al.; An effective vaccine against feline leukemia virus infection has been developed by the collection and concentration of tissue culture medium harvested from a tumor cell line . Lymphoid cells were grown to near saturation density in a normal growth medium and then transferred to a serum-free medium . The serum-free medium was collected, concentrated, and evaluated for its vaccine potential . Cats receiving the vaccine emulsified in complete Freund adjuvant developed high antiviral and antitumor titers and were protected (81%) against virus challenge . Cats receiving the vaccine without an adjuvant developed lower antibody levels and lower protection (53%) from viremia . Age-matched and litter-matched controls developed no antibody to test antigens before the challenge, and 100% became persistently viremic after the challenge . Vaccination with the soluble tumor cell antigen vaccine proved successful in preventing the induction of feline leukemia virus infection. J Biol Chem, 1981 Nov 10, 256(21), 10973 - 8 Phospholipid accumulation during the cell cycle in synchronous cultures of the yeast, Saccharomyces cerevisiae; Cottrell SF et al.; Phospholipid concentrations have been examined throughout successive cell cycles in synchronously growing cultures of the yeast, Saccharomyces cerevisiae . Total phospholipid phosphorus, as well as lecithin and phosphatidylethanolamine levels, exhibited stepwise increases during the cell cycle with step increments beginning just prior to new rounds of bud formation . Phosphatidylinositol and phosphatidylserine levels, on the other hand, showed what have been interpreted to be peak concentrations near the time of bud formation . Cardiolipin content varied considerably and was dependent upon the carbon source of the growth medium . Glucose-grown cells exhibited peak concentrations of cardiolipin near the time of bud formation, with marked decreases after this time . In contrast, galactose-grown synchronous cells exhibited stepwise increments in cardiolipin content, with step increases occurring near the time of new rounds of bud formation . Step or peak increases in cardiolipin, as well as all other phospholipids, were found to coincide with the time of stepwise increases in cytochrome c oxidase activity in these cells . No correlations were observed between the elaboration of mitochondrial membranes during the synchronous cell cycle and the observed patterns of phospholipid increase. Mikrobiologiia, 1981 Nov-Dec, 50(6), 1046 - 52 {Effect of the age of the culture and the composition of the medium on alkaloid biosynthesis by Penicillium gorlenkoanum}; Kozlovskii AG et al.; The production of the alkaloids costaclavine and epicostaclavine by Penicillium gorlenkoanum was studied as a function of the culture age and the composition of the growth medium . The alkaloids were found in the mycelium and, in great quantities, in the cultural broth . The production of the extracellular alkaloids started from the first days of growth and run in parallel to the biomass accumulation; the synthesis of costaclavine and epicostaclavine was maximal at the end of the logarithmic-stationary growth phases . The medium supplied with mannitol, succinic acid and 1% KH2PO4 was optimal for epicostaclavine synthesis . A carbohydrate or organic acid substitution as well as a variation in the concentration of phosphate changed the proportion between epicostaclavine and costaclavine contents . The biosynthesis of epicostaclavine was inhibited almost entirely by the glucose deficiency (2%) in the medium . This glucose concentration, phosphate excess (10 g/l), and the presence of citric or tartaric acids created favourable conditions for costaclavine biosynthesis. Mikrobiologiia, 1981 Nov-Dec, 50(6), 1042 - 5 {Comparative physiological and biochemical characteristics of Aspergillus niger isolated from the mesosphere and of its mutant}; Imshenetskii AA et al.; The aim of this work was to study certain physiological-biochemical characteristics of Aspergillus niger, strain 26, isolated from the mesosphere as well as those of its mutant having light-brown conidia . The parent strain and its mutant were grown in a liquid Chapek medium to study accumulation of the biomass, changes in the pH of the medium, as well as assimilation of glucose, nitrogen (NO3-) and phosphorus (PO4-) . The content of polysaccharides, protein, RNA and DNA was determined in the biomass . The parent culture and its mutant had the same growth dynamics and changes in the pH of the growth medium . They assimilated nitrogen, phosphorus and glucose at the same rate . No significant differences were found in the content of DNA, RNA, protein and polysaccharides . Lipids were an exception: their content was higher by ca . 26% in the mutant as compared with the parent strain . Apparently, the elevated sensitivity of the mutant to UV is due not only to a loss of certain pigments, but also to a damage of other protective mechanisms of the cell. Mol Cell Biol, 1981 Nov, 1(11), 1007 - 15 Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae; Kief DR et al.; We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus . The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source . The results demonstrated that ribosome content is proportional to growth rate . Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins . When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with {methyl-3H}methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min . Labeling with {3H}leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins . Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min . The data suggest that S . cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins. Br J Ind Med, 1981 Nov, 38(4), 394 - 6 Cotton bacterial endotoxin assessed by electron microscopy; Helander I et al.; A piece of bale cotton was incubated in nutrient broth . Electron microscopic inspection of the cotton and the broth showed Gram-negative bacteria with long flagella, loosely attached to the cotton fibres . Large amounts of endotoxin liberating from these bacteria were visible in the growth medium. Cancer Res, 1981 Nov, 41(11 Pt 1), 4493 - 8 Effects of deoxynucleosides on cultured human leukemia cell growth and deoxynucleotide pools; Ross DD et al.; We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd) . Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar . Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium . The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM . For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM . Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10) . In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively . Incorporation of {3H}dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo . Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance {3H}dCyd incorporation in either HL-60 or K-562 cells . Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning . These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself. Cancer Res, 1981 Nov, 41(11 Pt 1), 4579 - 87 Induction of the regulatory subunit of type I adenosine cyclic 3':5'-monophosphate-dependent protein kinase in differentiated N-18 mouse neuroblastoma cells; Liu AY et al.; The expression of a adenosine cyclic 3':5'-monophosphate (cAMP)-binding protein, regulatory subunit of the type I cAMP-dependent protein kinase (Rl), and its functional significance in the differentiation of N-18 mouse neuroblastoma cells were examined . 8-Azidoadenosine cyclic 3':5'-{32P}monophosphate, a photoaffinity-labeling analog of cAMP, and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to identify and quantitate cAMP-binding proteins in cell extracts . The induction of differentiation of N-18 mouse neuroblastoma cells, initiated either by adding dibutyryl adenosine cyclic 3':5'-monophosphate to the growth medium or by culturing cells in medium supplemented with 1% fetal calf serum, led to a 3-fold increase in the amount of 8-azidoadenosine cyclic 3':5'-{32P}monophosphate incorporated into Rl, when assayed in vitro . This increased incorporation was attributable to an increase in the amount of Rl rather than to an increase in the affinity of Rl for 8-azidoadenosine cyclic 4':5'-{32P}monophosphate . The subunit molecular weight, isoelectric point, and immunoreactivity of Rl were found to be identical to that of the regulatory subunit of the type I cAMP-dependent protein kinase purified from bovine skeletal muscle . The increase in Rl was not accompanied by an increase in the cAMP-dependent protein kinase activity . DEAE-cellulose column chromatography confirmed the induction of Rl as a free cAMP-binding protein in the differentiated neuroblastoma cells . The possibility of a growth-dependent regulation of Rl was also examined . Addition of 2% dimethyl sulfoxide to cultures of N-18 mouse neuroblastoma cells inhibited cell growth without increasing the specific activity of Rl . Dimethyl sulfoxide had no effect on neurite outgrowth or acetylcholinesterase activity, two parameters characteristic of differentiated cells . The fact that the induction of Rl coincided with differentiation of the neuroblastoma cells suggests that the expression of Rl may be used as a biochemical index of differentiation in these cells . The presence of a free cAMP-binding protein, not associated with cAMP-dependent protein kinase in neuroblastoma cells, raises important considerations concerning the action of cAMP in the regulation of growth and differentiation. J Cell Physiol, 1981 Nov, 109(2), 309 - 15 Growth of C6 glioma cells in serum-containing medium decreases beta-adrenergic receptor number; Dibner MD et al.; Rat C6 glioma cells were grown in 5% fetal bovine serum-containing medium and under serum-free, defined conditions . In order to ask whether cells grown in serum-free medium are phenotypically identical to cells grown in serum, we examined effects of cell growth under both conditions on the beta-adrenergic receptor, a cell surface marker that activates adenylate cyclase and thereby regulates these cells . beta-Adrenergic receptors were qualitatively similar in cells grown in serum-containing and serum-free media, but the number of receptors was 30-50% less in cells grown with serum . This effect required several days to occur or to be reversed . The decreased number of receptors appeared to be caused by an inhibitory effect of serum on receptor number and not by a stimulatory action of the constituents of the serum-free medium . Growth medium with 5% fetal bovine serum maximally inhibited beta-adrenergic receptor numbers with 1% serum causing a half-maximal inhibition . The ability of serum to inhibit the expression of beta-adrenergic receptors could be blocked by dialyzing but not by boiling fetal bovine serum . beta-Adrenergic receptor-stimulated cyclic AMP accumulation was also decreased by growth in medium containing serum . These studies demonstrate that compared to growth of cells in serum-free medium, growth in serum-containing medium can inhibit expression of cell surface beta-adrenergic receptors . These results imply that the presence of serum in medium in which cells are grown alters properties in the plasma membrane and may alter hormonal responses in such cells. Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6907 - 11 Epibolin: a protein of human plasma that supports epithelial cell movement; Stenn KS; Earlier studies suggested that there is a specific activity in mammalian serum and plasma that supports epidermal (epithelial) cell movement . This activity was shown to be nondialyzable and heat labile . In the studies reported here, using standard biochemical procedures--i.e., ammonium sulfate fractionation, ion-exchange and gel filtration chromatography, isoelectric precipitation, and preparative polyacrylamide gel electrophoresis--we have purified a factor from human plasma that supports epidermal cell movement . The factor travels as an apparent single band on disc gel electrophoresis and corresponds to a glycosylated single-chain protein of approximately 65,000 +/- 3,000 daltons . The purified fraction is necessary and sufficient for dissociated epidermal cells, (ii) outgrowth of epithelial sheets from skin explants, and (iii) epiboly, epithelial sheet movement over a floating skin explant . The purified fraction is active at a concentration of 1-2 micrograms/ml of growth medium . It is destroyed by trypsin and its activity is augmented more than 10-fold by a second, as yet unpurified, fraction of plasma . These studies support the notion that a single protein of plasma supports epidermal cell movement and that this protein may play an important role in wound closure . Because it supports epiboly, the most biologically relevant of the assays, it has been named epibolin. J Biol Chem, 1981 Oct 25, 256(20), 10623 - 7 The mannose 6-phosphate receptor of Chinese hamster ovary cells . Compartmentalization of acid hydrolases in mutants with altered receptors; Robbins AR et al.; The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A . R., Myerowitz, R., Youle, R . J., Murray, G . J., and Neville, D . M., Jr . (1981) J . Biol . Chem . 256, 10618-10622) . Cells were grown in the presence of {3H}mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography . About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15% . The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity . Intracellular levels of these enzymes were reduced in the mutants . The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments . {35S}methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate . Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor. Biochim Biophys Acta, 1981 Oct 12, 677(2), 269 - 73 Constitutive behavior of methionyl-tRNA synthetase compared to repressible behavior of methionine adenosyltransferase in mammalian cells; Rubnitz JE et al.; Methionine adenosyltransferase, one of the two major enzymes utilizing methionine, is regulated by the levels of methionine in the growth medium (Jacobsen, S.J., Hoffman, R.M . and Erbe, R.W . (1980) J . Natl . Cancer Inst . 65, 1237--1244, and Caboche, M . and Mulsant, P . (1978) Somatic Cell Genet . 4, 407--421) . We report here that methionyl-tRNA synthetase, unlike methionine adenosyltransferase, behaves in a constitutive manner with respect to the concentration of methionine in the culture medium . This behavior is seen in Chinese hamster ovary cells and in normal diploid and SV40-transformed human fibroblasts . Although the kinetics of regulation of methionine adenosyltransferase and methionyl-tRNA synthetase by exogenous methionine are clearly different, the levels of the two enzymes in the human cell lines are similar. Gann, 1981 Oct, 72(5), 717 - 22 Effect of prolonged exposure of HELA S3 cells to low concentrations of misonidazole; Ohizumi Y et al.; The cytotoxic and radiosensitizing effects of low concentrations of misonidazole were examined by measurement of the colony-forming ability and growth of HeLa S3 cells . Under hypoxic conditions, cytotoxicity depended on the misonidazole concentration and the exposure time . After 24 hr exposure, the surviving fraction of cells treated with 0.1 mM misonidazole decreased to 0.2 . The ability of cells to adhere to the growth surface of plastic dishes was reduced by 0.1 mM misonidazole upon 24 hr hypoxia and this effect persisted even during subsequent euoxia, irrespective of whether the drug was removed from the original growth medium . The growth of aerobic cells was not affected when 0.1 mM misonidazole containing medium, obtained from the hypoxia-treated dishes, was applied, nor when those cells were exposed for 72 hr to freshly prepared 1.0 mM misonidazole . Upon 24 hr exposure, 0.1 mM misonidazole exerted no obvious radiosensitizing effect on hypoxic cells . Based on these results, it is suggested that continuous administration of low doses of misonidazole may be efficacious as a specific chemotherapeutic in the treatment of hypoxic cells in tumor tissue. Cancer Res, 1981 Oct, 41(10), 4093 - 100 Growth of human mammary epithelial cells on collagen gel surfaces; Yang NS et al.; We have developed a method for sustained growth of human mammary epithelial cells in monolayer cultures . Epithelial organoids derived from solid breast tissues were grown on the surface of thin (approximately 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum, and horse serum . To transfer the cultures, the collagen layers were dislodged and digested with collagenase . The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylene-diaminetetraacetate . Dissociated single cells were repleted with 75 to 95% efficiency onto collagen layers or tissue culture plastic surfaces . The dissociated cells could also be cryopreserved and reactivated with greater than 80% plating efficiency on collagen layers . Normal human mammary epithelial cells grown under these conditions progressed through 12 to 15 population doublings . The population-doubling times for normal and malignant mammary cells on collagen layers were 34 and 65 hr, respectively . After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen . Within the gels, the cells became organized into three-dimensional tubular structures . The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation, and cell transfer from monolayers. J Cell Physiol, 1981 Oct, 109(1), 171 - 9 Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan; Didinsky JB et al.; It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins . These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential . We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth . Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced . At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated . We determined cumulative population doublings and also estimated the number of cell generations attained under each condition . FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan . The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used . The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical. J Cell Physiol, 1981 Oct, 109(1), 1 - 15 Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin; Rohrlich ST et al.; Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin . Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets . It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction . This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS) . Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS . The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods . It is not labeled metabolically with 3H-leucine . Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing . The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator . They are immunologically indistinguishable . The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum . These molecules may serve to protect HuEL cells against proteases they generate. Biull Eksp Biol Med, 1981 Oct, 91(10), 460 - 2 {Interferons produced by the cells of newborn mice in the presence of sera from newborn and adult animals}; Malinovskaia VV et al.; Fibroblasts of newborn mice produced far less amount of interferon in the presence of sera from newborn animals than in the presence of sera from adult animals . The interferons obtained were purified by adsorption chromatography on porous glass and were analyzed by electrophoresis in polyacrylamide gel . It has been shown that antiviral activity of interferon preparations obtained in the presence of sera from newborn mice was associated with the fraction of 45 Kd . Addition into the growth medium of sera from adult animals led to the production by the same cells of interferon activity associated with 41 and 28 Kd fractions . It is assumed that the sera of newborn mice contained the components influencing the molecular content of interferon produced by the cells of newborn animals. Hoppe Seylers Z Physiol Chem, 1981 Sep, 362(9), 1219 - 27 {Degradation of L-phenylalanine and of aromatic carboxylic acids by chloridazon-degrading bacteria . Combination of side chain degradation and dioxygenase pathway}; Wegst W et al.; Strain N of Chloridazon-degrading bacteria degrades phenylalanine via cis-2,3-dihydro-2,3-dihydroxyphenylalanine,2,3-dihydroxyphenylalanine aspartate and 4-hydroxy-2-oxovalerate {Hoppe-Seyler's Z . Physiol . Chem . 360, 957--969, (1979); Biochem . J . 194, 679--684 (1981)} . cis-2,3-Dihydro-2,3-dihydroxyphenylalanine and 2,3-dihydroxyphenylalanine as well as phenylpyruvate, cis-2,3-dihydro-2,3-dihydroxyphenylpyruvate, 2,3-dihydroxyphenylpyruvate, cis-2,3-dihydro-2,3-dihydroxyphenylacetate, 2,3-dihydroxyphenylacetate and 2,3-dihydroxybenzaldehyde are detectable in the medium of strain E during growth on phenylalanine . Incubation with phenylacetate, 3-phenylpropionate or 4-phenylbutyrate leads to the accumulation of the corresponding cis-2,3-dihydro-2,3-dihydroxyphenyl derivatives . These compounds are transformed with dihydrodiol dehydrogenase to 2,3-dihydroxyphenylacetate, 3-(2,3-dihydroxyphenyl)propionate and 4-(2,3-dihydroxyphenyl)-butyrate, 3-(2,3-dihydroxyphenyl)propionate is attacked by a catechol 2,3-dioxygenase and the meta-cleavage product is again cleaved by a hydrolase yielding succinate . In a similar reaction sequence the degradation of 4-phenylbutyrate leads to the formation of glutarate . From the growth medium of strain E on phenylacetate also small amounts of 2-, 3- and 4-hydroxyphenylacetate were isolated . Resting cells were shown to metabolize 3- and 4-hydroxyphenylacetate via homogentisate and 3,4-dihydroxyphenylacetate . In the culture medium of strain K2AP benzoate could be detected . Pathways for the degradation of phenylalanine and aromatic carboxylic acids in chloridazon degrading bacteria are proposed. J Virol, 1981 Sep, 39(3), 903 - 13 Characterization of intracellular and extracellular vaccinia virus variants: N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine interferes with cytoplasmic virus dissemination and release; Hiller G et al.; Infectious vaccinia virus can be purified from whole cells by experimentally induced lysis (intracellular virus) or from supernatant growth medium (extracellular virus) . Extracellular virus and intracellular virus differed by buoyant density (1.237 versus 1.272 g/cm3), phospholipid content and composition, and polypeptide pattern . Differences in structural polypeptides on the virus surface could be detected by lactoperoxidase-catalyzed radioiodination or Brij treatment . Characteristic of extracellular virus was an additional polypeptide, with a molecular weight of 37,000 (37K), which represented 5 to 7% of the total particle protein . Antibodies to the 37K protein detected only some of the cell-associated particles late in normal infection . Upon treatment of infected cultures with N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine, a drug which prevents vaccinia virus release, no particle-associated 37K protein could be detected . In all other properties tested so far, except for a slight difference in phospholipid composition, the virus obtained in the presence of the drug resembled the normal intracellular virus . N1-Isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine prevented vesicularization of intracellular viral particles . Lack of vesicularization was accompanied by the absence of particle-associated 37K viral protein and seemed to correlate with an inhibition of virus dissemination to the cell periphery. Chem Biol Interact, 1981 Sep, 36(3), 259 - 74 Binding of methylmercury(II) by HeLa S3 suspension-culture cells: intracellular methylmercury levels and their effect on DNA replication and protein synthesis; Gruenwedel DW et al.; HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using 203Hg-labeled methylmercuric chloride as radioactive marker . Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells . The results show that methyl-mercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells . The amounts of bound methylmercury, {CH3Hg(II)}bound, given in mol/cell, range from 2 X 10(-16) (at 1 h of incubation and at 1 microM CH3Hg(II) in the medium) to almost 4 X 10(-14) (at 24 h of incubation and at 100 microM CH3Hg(II) in the medium) . A {CH3Hg(II)}bound value of about 30 X 10(-16) mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place . Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium . Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 X 10(4) l/mol and for the maximum concentration of cellular binding sites the value 2.40 X 10(-14) mol/cell or 1.45 X 10(10) sites/cell . Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too . The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place . Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution. Cancer Res, 1981 Sep, 41(9 Pt 1), 3592 - 6 Growth and differentiation of human and murine erythroleukemia cell lines in serum-free synthetic medium; Pessano S et al.; Only two chemicals (transferrin and selenium dioxide) are required to supplement serum-free Roswell Park Memorial Institute Medium 1640 for long-term growth and for spontaneous and induced differentiation of established lines of human and mouse erythroleukemia cells . We describe here two serum-free media (a minimal synthetic medium and a high-density synthetic medium) that support the growth and differentiation of human K562(S) and mouse clones 745, 707, and 3TCl 12 erythroleukemia cell lines in long-term culture . The doubling times of the erythroleukemic cell populations are longer in minimal synthetic medium that in serum-containing medium . Cell saturation density in minimal growth medium is one-half that obtained in serum-containing medium for clone 745, whereas for K562(S) it is approximately the same . Cell saturation density in high-density medium (containing albumin) is greater than that achieved in serum-containing medium for K562(S), whereas for clone 745 cell saturation density increases for cells in midlogarithmic growth, although not to the density of cells grown in serum-containing medium . The differences in saturation density are due to a decreased doubling time as well as to better survival of the cells 3 or 4 days after plating . The cells can grow in the synthetic media and be passaged for as many generations as desired without impairment of growth capabilities . In the minimal synthetic medium, spontaneous differentiation of erythroleukemia cells continues to occur, indicating that spontaneously differentiating cells are the result of intracellular mechanisms controlling the expression of a genetic program of some of the cells at any given time . Hemoglobin synthesis can be induced in cells growing in synthetic medium by using lower concentrations of the same inducers that are effective in serum-containing medium, indicating that these chemicals do not depend on serum factors to initiate the process of differentiation . The percentage of benzidine-positive cells and the concentration of hemoglobin per cell, however, are less in the synthetic medium than in serum-containing medium, suggesting that serum factors do play a role in modulating the extent of hemoglobin synthesis . The types of hemoglobins synthesized by cells in synthetic medium are identical to those reported in serum-containing medium. Cell, 1981 Sep, 25(3), 773 - 82 Growth-rate-dependent regulation of ribosome synthesis in E . coli: expression of the lacZ and galK genes fused to ribosomal promoters; Miura A et al.; Hybrid transducing phages were constructed in vitro that carry the galK gene fused to each of three ribosomal promoters: the promotor for an rRNA operon (rrnE); the promoter for the spec r protein operon and the promotor for the alpha r protein operon . We also constructed hybrid transducing phages that carry the IacZ gene fused to the promoter for the rrnE operon or to the promoter for the spc r protein operon . The amounts of galactokinase (or beta-galactosidase) were analyzed in lysogens carrying these various transducing phages grown in several different growth media . The synthesis rate of galactokinase (or beta-galactosidase) from the fused rrn-gal (or rrn-lac) operon relative to the total protein synthesis rate increased with increasing growth rate, as expected from the transcriptional activity of rRNA operons . In contrast, the relative synthesis rate of galactokinase (or beta-galactosidase) from the operon fused to alpha or spc r protein promoter remained approximately constant with increasing growth rate . These results were interpreted to mean that the characteristic increase in the relative synthesis rate of r protein with increasing growth rate is determined not by transcription regulatory mechanisms, but by posttranscriptional mechanisms, which presumably involve the feedback inhibition of r protein mRNA translation by free r proteins. J Neuropathol Exp Neurol, 1981 Sep, 40(5), 512 - 25 Growth characteristics of human glioma-derived and fetal neural cells in culture; Icard C et al.; Growth characteristics of human fetal neural cells (CH) and human glioblastoma multiforme-derived cells (12-18) in culture were compared . Cells were grown to confluent densities of 38,000 to 42,500 cells/cm2 for CH and 85,800 to 87,100 for 12-18 . Population doubling times were 40.0 +/- 5.1 hr and 66.5 +/- 9.8 hr for CH and 12-18 cells, respectively . The mean DNA content per cell of the glioma-derived cells was twice that of the fetal brain cells at sparse, log, and confluent cell densities . High concentrations (40%) of serum in growth medium increased DNA contents in confluent CH, but not 12-18, cells . The amount of protein per cell also was consistently higher in glioma cells than CH cells, but, as cell densities increased, protein contents decreased for both: 1200 to 700 pg/cell in glioma cells, and 840 to 560 pg/cell in CH cells . In each cell line, initial rates of {3H}ThdR incorporation into TCA precipitable material decreased as cell density increased, but confluent glioma-derived cells incorporated 10 times more {3H}ThdR than confluent fetal cells . Almost all CH cells had a normal diploid chromosome number of 46 . A histogram showing the relative frequencies of chromosome numbers of glioma-derived cells had peaks of 52, 79, and 105 chromosomes per metaphase, indicating a haploid number of 26 for most cells . Lengths of cell cycle phases, determined using autoradiographic techniques, indicate that glioma-derived cells had a longer generation time and S period than fetal neural cells . These data demonstrate several biological differences between glioblastoma-derived cells and non-neoplastic fetal neural cells, indicating that this system is of potential value for comparative studies on growth control and contact inhibition. Acta Virol, 1981 Sep, 25(5), 257 - 65 Resistance of Semliki forest virus protein synthesis to high salt treatment; Erdei S; Selective translation of Semliki Forest virus-specific mRNA occurred in virus-infected cells exposed to hypertonic growth medium . The selective resistance of the virus-specific protein synthesis could be detected at a wide NaCl concentration range and was more significant at lowered incubation temperature (28 degrees C) . It is suggested that the translation of the structural proteins encoding subgenomic 26 S RNA is more resistant to the hypertonic initiation block than the translation of the genomic 42 S RNA which codes for the non-structural viral proteins. Biochemistry, 1981 Sep 1, 20(18), 5336 - 40 Biosynthesis of o-succinylbenzoic acid in a men- Escherichia coli mutant requires decarboxylation of L-glutamate at the C-1 position; Meganathan R et al.; A men- mutant of Escherichia coli, AN 209, which accumulates o-succinylbenzoic acid, has been used for a direct study of the biosynthesis of this benzenoid compound . Samples of labeled glutamic acids were added to growth media, and the o-succinylbenzoic acid was isolated and converted to a dimethyl derivative . This dimethyl derivative was purified on thin-layer chromatograms and by gas chromatography . When the glutamic acid used as precursor contained 14C at position 5, or was uniformly labeled, the dimethyl o-succinylbenzoate contained radioactivity (as shown by radiogas chromatography) . However, from {1-14C}glutamate, the dimethyl o-succinylbenzoate was without radioactivity . Hence, in the biosynthesis of o-succinylbenzoate, carbon atom 1 of glutamate is lost, and carbon atoms 2-5 are retained . It was also shown that this mutant lacked the enzyme dihydroxynaphthoic acid synthase . It should, therefore, continue to be classified as a menB mutant, rather than as a member of the newly created menE group (lacking o-succinylbenzoate-CoA synthetase). Brain Res, 1981 Aug 31, 219(2), 407 - 21 Growth of adult superior cervical ganglion explants in serum-free media; Dombrowski AM et al.; Neurite outgrowth from explants of superior cervical ganglion from adult rats can be achieved in a serum-free medium . Extensive neurite outgrowth occurred from ganglion explants maintained in Eagle's minimum essential medium supplemented with either 10% (V/V) fetal calf serum or 1% (W/V) bovine serum albumin and nerve growth factor . After one week in culture, the ATP content of explants maintained in the serum-free medium was slightly higher than that noted in explants cultured in the presence of fetal calf serum and amounts of phosphocreatine were significantly lower . Despite these differences in high energy phosphate content, the abundance and morphology of neuritic outgrowth were essentially the same from explants cultured in the two types of media . Comparable activities of a number of NADP+-dependent dehydrogenases were noted in explants maintained in the two types of media . Increases in the activities of the oxidative enzymes of the pentose pathway, which occur in axotomized ganglia in vivo, were observed in the cultured ganglion explants . NADP+-dependent isocitrate dehydrogenase activity remained constant in ganglion explants in vitro, and measurements of this activity were employed in a new method to quantitate neurite outgrowth . The activity of isocitrate dehydrogenase in lyophilized neurite processes that had grown out onto a Millipore filter substrate correlated well with visual estimates of neuritic outgrowth . Substitution of delipidated for normal bovine serum albumin in the growth medium resulted in a significant decrease in neuritic outgrowth from ganglion explants from both adult and weanling rats . Addition of fatty acids to media containing delipidated bovine serum albumin enhanced neuritic outgrowth in explants of weanling rats . Thus, lipophilic substances bound to bovine serum albumin including fatty acids appear necessary for optimal growth of neurites from explants of the rat superior cervical ganglion. Biochim Biophys Acta, 1981 Aug 17, 676(2), 221 - 5 High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis; Goldman ME et al.; Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine . In addition, small amounts of epinephrine and norepinephrine were detected . Logarithmic-phase strain NT-1 cells contained 249 +/- 44 pg dopamine/10(6) cells compared to 477 +/- 42 pg/10(6) cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells . The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells . There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells. Biochim Biophys Acta, 1981 Aug 13, 660(2), 371 - 4 Regulation of enterochelin synthetase in Escherichia coli K-12; Greenwood KT et al.; Enterochelin synthetase activity is controlled by both repression and feed-back inhibition mechanisms . Inclusion of iron in growth media results in synthesis of all four (D, E, F and G) components of enterochelin synthetase being repressed . The specific inhibition of L-serine activation (partial reaction catalyzed by the F component) by the end products, ferric-enterochelin and 2,3-dihydroxybenzoylserine, is shown to inhibit overall enterochelin synthetase activity. Biochim Biophys Acta, 1981 Aug 5, 676(1), 91 - 100 Cyclic nucleotide metabolism in differentiated and undifferentiated epithelial thyroid cells in culture; Mandato E et al.; A highly differentiated thyroid cell line (FR-RL) was compared with a less differentiated (FR-T Cl1) and an undifferentiated (1-5G) cell line . FR-TL is modulated in vivo and in vitro by thyrotropin and has the lowest adenylate cyclase and guanylate cyclase and the highest phosphodiesterase activities . In contrast, 1-5G tumor cells do not respond to thyrotropin and have the highest adenylate cyclase guanylate cyclase and lowest hydrolyzing enzyme activities . Intermediate enzyme activities were found in FR-T Cl1 cells . The differences between the two normal rat thyroid cell lines are not due to differences in the composition of the growth medium. J Invest Dermatol, 1981 Aug, 77(2), 221 - 4 Induction of lymphocyte differentiation by epidermal cultures; Rubenfeld MR et al.; Human and murine lymphoid cell populations were induced to express terminal deoxynucleotidyl transferase, a marker of early lymphoid differentiation, by exposure to allogeneic or syngeneic epidermal cells . Control growth medium, fibroblasts, or a mammary epithelial cell line did not induce this marker . These findings suggest that epidermal cells can induce lymphoid cell differentiation in vitro. Br J Cancer, 1981 Aug, 44(2), 219 - 35 Cytotoxic and clastogenic effects of soluble and insoluble compounds containing hexavalent and trivalent chromium; Levis AG et al.; Cr(III) and Cr(VI) compounds of varying solubilities have been tested in vitro for their ability to inhibit cell growth and nucleic acid and protein syntheses in BHK cells, to induce alterations in the mitotic cycle in HEp cells, and to increase the frequency of chromosomal aberrations and sister chromatid exchanges (SCE) in CHO cells . All Cr(VI) compounds, and particularly those containing soluble Cr(VI), such as potassium dichromate and zinc yellow, differentially inhibit macromolecular syntheses in BKH cells, that of DNA being always the most affected . Among Cr(III) compounds, which generally have very low cytotoxicity, chromite is particularly active, and inhibits cell growth and DNA synthesis even more than the poorly soluble Cr(VI) compounds . Preincubation in growth medium, with or without metabolizing cell cultures, solubilizes considerable amounts of Cr(VI) from zinc yellow and chromite, but significant amounts are also obtained from the most insoluble Cr(VI) pigments . When BHK cells are treated with such preincubated solutions, reduction of soluble Cr(VI) to Cr(III) by cell metabolites is seen with all Cr(VI) compounds, accompanied by decreased cytotoxicity . The same differences between Cr(VI) and Cr(III) compounds apply to the cytotoxic effects on mitosis of HEp cells and the clastogenic effects on CHO cells . The activity of chromite, the only Cr(III) pigment capable of significantly increasing the frequency of SCE, is due to contamination with soluble Cr(VI) . In contrast to the very low cytotoxicity of Cr(III), much higher chromium levels are detected in the cells incubated with soluble Cr(III) than with the same concentrations of soluble Cr(VI) . 50% and 75% of chromium accumulated in the cells during treatments with Cr(VI) and Cr(III) respectively remains firmly bound to the cells, even when they are incubated for up to 48 h in normal growth medium . Chromium accumulated in the cells after treatment with Cr(III) is most probably bound to the cell membrane, whereas some of the Cr(VI) is transported through the cell membrane and reduced in the cell nucleus . The results of the present investigation are in agreement with those obtained with the same Cr(VI) and Cr(III) compounds in mutagenicity assays in bacteria and carcinogenicity tests in rodents . A re-evaluation of the mechanisms of chromium carcinogenisis is proposed. Am J Hosp Pharm, 1981 Aug, 38(8), 1144 - 7 Effect of laminar air flow and clean-room dress on contamination rates of intravenous admixtures; Brier KL et al.; The effect of laminar air flow conditions and clean-room dress on the microbial contamination rates of intravenous admixtures was investigated . Intravenous admixtures were prepared by one investigator using aseptic technique under four environmental conditions: laminar air flow conditions with clean-room dress; laminar air flow without clean-room dress; clean table top with clean-room dress; and clean table top without clean-room dress . In each environmental condition, 350 admixtures were compounded . Negative-control samples (n = 150) were also tested, as were 10 positive-control samples . Samples were tested in each of two growth media and incubated at 35 degrees C for 14 days or until growth occurred . The incidence of contamination of admixtures compounded in laminar air flow conditions was significantly less than the contamination of those compounded on a clean table top (p less than 0.05) regardless of the operator's dress . The incidence of contamination of admixtures compounded while wearing clean-room dress was not significantly different from those prepared while not wearing clean-room dress regardless of the environment in which the admixture was prepared . The overall low level of contamination {0.79% (11/1400)} was inconclusive regarding the effect of dress on the incidence of contamination when admixtures were prepared under LAF conditions . It is concluded that, when one adheres to aseptic technique, the environment in which admixtures are compounded is the most important variable affecting the microbial contamination rate. J Bacteriol, 1981 Aug, 147(2), 373 - 81 Dihydroxy and monohydroxy fatty acids in Legionella pneumophila; Mayberry WR; Five strains of Legionella pneumophila were examined for the presence of hydroxy fatty acid . The cellular distribution of the fatty acids was also determined, as was the variation of hydroxy acid production on five growth media . The strains tested all produced approximately 5 mol% of hydroxy fatty acid, most of which was found in the nonextractable, alkali-stable, acid-labile (wall-associated, amide-linked) fraction . Three major hydroxy acids were found, along with several minor components . The major hydroxy acids were analyzed by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and infrared spectrophotometry . These compounds were tentatively identified as 3-hydroxy-12-methyltridecanoate, 3-hydroxy-n-eicosanoate, and a novel dihydroxy acid, 2,3-dihydroxy-12-methyltridecanoate . The total amount of hydroxy acid produced, as well as the profile of the hydroxy acids, remained relatively unchanged with respect to strain and growth medium. Appl Environ Microbiol, 1981 Aug, 42(2), 200 - 3 Serum substitute in epithelial cell culture media: nonfat dry milk filtrate; Fassolitis AC et al.; A number of milk types and milk fractions were investigated as possible substitutes for serum in cell culture media . A filtrate of reconstituted nonfat dry milk showed promise . Culture fluids containing 5% of the nonfat dry milk filtrate were used to propagate primary and continuous cell cultures, and the cell growth from these cultures was compared with that of cells grown in a serum-containing medium . The nonfat dry milk filtrate-supplemented medium supported the growth of all epithelial cells tested, but two fibroblast-type cultures failed to replicate . Cells grown in the medium containing the milk filtrate grew slowly for 2 to 3 days and then propagated to confluency in 6 to 8 days . Viable cell counts of 9 days were comparable to those of serum-grown cells that had been propagated for 7 days . Cells grown in the milk filtrate could be split 1 to 4 when subcultures were prepared . Cell growth could be stimulated by refeeding on days 2 to 3 or by the addition of 30 microM 2-mercaptoethanol to the growth medium . Virus susceptibility and titer comparisons with poliovirus 1, coxsackievirus B2, echovirus 7, and herpes simplex virus indicated that approximately the same data were obtained when either the nonfat dry milk filtrate-treated or the serum-treated cells were studied . The nonfat dry milk filtrate is inexpensive, is easily prepared, and is a substitute for serum in epithelial cell culture media. J Biol Chem, 1981 Jul 25, 256(14), 7628 - 37 The cyclic nucleotide phosphodiesterase inhibitory protein of Dictyostelium discoideum . Purification and characterization; Franke J et al.; We have purified the glycoprotein inhibitor of the extracellular cyclic nucleotide phosphodiesterase of Dictyostelium discoideum to apparent homogeneity . The inhibitor has a molecular weight of 47,000 measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The interaction of the inhibitor and the cyclic nucleotide phosphodiesterase occurs with 1:1 stoichiometry and with a dissociation constant of about 10(-10) M . Periodate oxidation of the inhibitor or of the enzyme destroys concanavalin A binding ability but does not affect the formation of the enzyme-inhibitor complex . Inhibitor is not produced by cells during logarithmic growth but appears in quantity during stationary phase and after transfer from growth medium to phosphate buffer. I