Med Biol, 1984, 62(6), 338 - 43 Bioluminescence of cellular ATP: a new method for evaluating cytotoxic agents in vitro; Kangas L et al.; Rat Walker 256 carcinosarcoma, human MCF-7 cell line and a specimen of ovarian serous cystadenocarcinoma were cultured in vitro and exposed to different cytostatic drugs . The drug effects were evaluated by bioluminescence, i.e., by measuring the levels of adenosine triphosphate (ATP), the basic energy source of the living cells . The intracellular ATP was released by TCA or NRS -reagent, and the ATP levels were measured directly from an aliquot of the growth medium without any extraction or precipitation steps . ATP level was significantly correlated with cell number, viability, {3H}-thymidine incorporation and stem cell assay . The most important advantages of bioluminescence method was speed, technical simplicity and good sensitivity (about 500 cells/sample easily quantitated, the results are seen directly within a few seconds) and flexibility (any cell line and drug may be studied by many different test designs) . ATP method obviously describes the "well- being" of the cultured cells . According to our experience, the ATP-bioluminescence method is a powerful alternative to any other cell growth estimation method in vitro . It can be used especially in primary screening of the cytostatic activity of any known or unknown substance as well as in attempts to select an individual drug therapy for patients with cancer. J Membr Biol, 1984, 82(1), 59 - 65 Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: the nature of the signal; Moran A et al.; We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK1 . Assayed in physiological salt solution, the ratio of the concentration of alpha-methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium . In this study, using a cloned line of LLC-PK1 cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa . By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1 mM glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium . Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter . Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism. Bioelectromagnetics, 1984, 5(2), 117 - 29 System for the exposure of cell suspensions to power-frequency electric fields; Kaune WT et al.; A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line . In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated . Electric fields in the range 0.02-3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5% . The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 microT . The temperatures of the experimental and control cell suspensions are held fixed within +/- 0.1 degrees C by a water bath . Special chambers were developed to hold cell cultures during exposure and sham exposure . Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16-17 h, approximately the same as for CHO cells grown under standard cell-culture conditions. Acta Biochim Pol, 1984, 31(4), 401 - 8 The possible occurrence of zinc in ribitol and sorbitol dehydrogenases from Mycobacterium sp . 279; Szumilo T et al.; The activity of polyhydric alcohol dehydrogenases in Mycobacterium sp . 279 was studied under limitation of zinc in the growth medium . It was found that the activity of ribitol and sorbitol dehydrogenases were markedly lowered and that of D-arabinitol dehydrogenase remained unchanged in the Zn2+-deficient cells . Other ions tested i.e., Co2+, Cu2+, Ni2+ and Mn2+ failed to substitute Zn2+ ions in their effect on the enzyme activities . The Zn2+-responsive enzymes were sensitive to the chelating agents (1,10-phenanthroline, 2,2'-dipyridyl), whereas D-arabinitol dehydrogenase was insensitive . The results indicate possible existence of a zinc component in the ribitol and sorbitol dehydrogenases from Mycobacterium sp . 279. J Neurosci Res, 1984, 12(2-3), 311 - 22 Metabolism and function of gangliosides in developing neurons; Dreyfus H et al.; Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis) . The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides . Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single-species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations . Incubation of neurons with N-acetyl-D-{U-14C}mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides . The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10(-8) M and 10(-5) M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+-provoked one is abolished; decrease of the spontaneous and K+-stimulated release of dopamine; no effect on the spontaneous release of GABA for 10(-8) M gangliosides but an increase of both spontaneous and K+-provoked release for 10(-5) M gangliosides . The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms. Adv Exp Med Biol, 1984, 177, 229 - 40 Differential effects of sodium selenite and methylmercury(II) on membrane permeability and DNA replication in HeLa S3 carcinoma cells: a preliminary report regarding the modification of organomercurial toxicity by selenium compounds; Gruenwedel DW; When viewed in terms of their concentration in the growth medium, sodium selenite and methylmercuric hydroxide--administered individually to HeLa S3 cells--are of equal efficacy in inhibiting DNA synthesis: the dose-response curves overlap and 50% residual DNA synthesis occurs at 6.13 microM of either chemical . A different picture, however, emerges if replication is expressed as a function of the actual amounts of toxicant bound per cell . Now, the dose-response curves do not overlap and Na2SeO3 is much more toxic than CH3HgOH: 50% inhibition of DNA replication exists at 5.37 X 10(-17) moles of Se bound per cell and at 3.63 X 10(-15) moles of Hg bound per cell . Further, selenite is taken up by the cells more slowly than methylmercury and its (limiting) cellular concentration is below that of the organomercurial . Lastly, much higher levels of selenite in the growth medium are required to bring about the same degree of membrane damage as the one caused by methylmercury . These differential effects may have a bearing on the observation, well-known but thus far unexplained, that selenite and methylmercury are strikingly less toxic to animals when administered simultaneously than they are when administered individually: selenium may counteract the membrane-destabilizing characteristics of methylmercury and it may retard its binding to the cells . Data on the inhibition of DNA synthesis have been obtained when selenite and methylmercury are administered simultaneously to HeLa S3 cells in varied molar ratios . Best mutual protection appears to exist when the two chemicals are present in equimolar amounts or when there is a slight excess of selenite. Crit Rev Microbiol, 1984, 11(2), 129 - 55 Lipopolymers, isoprenoids, and the assembly of the gram-positive cell wall; Reusch VM Jr; Several lines of evidence suggest that Gram-positive bacterial cell surface polymers are synthesized by stepwise addition of polymer subunits to an amphipathic acceptor . In the case of membrane-bound lipopolymers such as mannan and lipoteichoic acid, the finished product may be covalently linked to a lipid anchor . In the case of polymers that are transferred into preexisting cell wall, such as teichoic acid and peptidoglycan, two alternative fates might be possible: (1) transfer into wall with concomitant or later cleavage of the lipid anchor, with recycling of the lipid anchor or secretion of the lipid anchor into the growth medium, and (2) transfer into wall without cleavage of the lipid anchor, resulting in maintenance of the covalent relationship between lipid anchor and polymer chain . In the latter case, a close relationship should be established between the cell wall and the plasma membrane . A number of Gram-positive bacteria have been shown to be resistant to plasmolysis . Therefore, a model for the assembly of the Gram-positive cell wall is proposed which takes into account a role for lipopolymeric intermediates and which views the establishment of resistance to plasmolysis as the natural consequence of such a mechanism. Avian Dis, 1984 Jan-Mar, 28(1), 266 - 72 Improving the Mycoplasma gallisepticum and M . synoviae antigen yield by readjusting the pH of the growth medium to the original alkaline state; Lin MY et al.; The pH of Mycoplasma gallisepticum (MG) and M . synoviae (MS) growth medium was readjusted back to the original alkaline state when the pH reached 6.1 (for MG) and 6.7 (for MS), and the medium was reincubated until the pH of the medium returned to 6.7 to 6.9 . The result was that MG and MS antigen yields were 43 and 54% higher than yields obtained at the usual harvest time. Br J Cancer Suppl, 1984, 6, 103 - 6 Indications for an inducible component of error-prone DNA repair in yeast; Siede W et al.; In a thermoconditional mutant of mutagenic DNA repair (rev 2ts = rad 5-8) of Saccharomyces cerevisiae recovery of survival and mutation frequencies can be monitored by incubating UV-irradiated cells in growth medium at a permissive temperature (23 degrees C) before plating and a shift to restrictive temperature (36 degrees C) . Inhibition of protein synthesis with cycloheximide during incubation at permissive conditions blocks this REV 2 dependent recovery process in stationary phase rev 2ts cells, whereas it can be reduced but not totally abolished in exponentially growing cells . These results indicate a strict dependence on post-irradiation protein synthesis in stationary phase cells and argue for a considerable constitutive level and only limited inducibility in logarithmic phase cells . The UV inducibility of the REV 2 coded function in stationary phase cells could be confirmed by analysis of the dose-response pattern of the his 5-2 reversion: in stationary phase rev 2ts cells, the quadratic component of the biphasic linear-quadratic induction kinetics found at 23 degrees C, which is interpreted as the consequence of induction of mutagenic repair, is eliminated at 36 degrees C. Appl Environ Microbiol, 1984 Jan, 47(1), 75 - 9 Proteinases produced by pseudomonads isolated from sheep fleece; London CJ et al.; Fifty-nine pseudomonads isolated from sheep fleece were able to grow on a minimal salts medium with glycerol as the sole source of carbon and energy . Many of these isolates showed additional growth when collagen-based or wool-based substrates were included in the medium . After several days of incubation with these substrates, the nature of soluble proteins present in the growth medium was investigated by using polyacrylamide gel electrophoresis . Up to four major bands of protein with proteinase activity and with widely different electrophoretic mobilities were detected in the gels; one of the bands appeared as a doublet at times . The electrophoretic mobilities of each class of proteinase were similar for the different pseudomonads examined, but the proteinase (or combination of proteinases) induced depended on the protein substrate and strain or species of pseudomonad used. Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Jan, 45(1), 27 - 31 Growth in cadmium-containing medium induces resistance to heat in E . coli; Roberts PB; We have shown that 10 microM Cd2+ in the growth medium can induce resistance to subsequent heat treatment in E . coli B/r . Resistance was shown by cells during an extended lag phase and, especially, during log phase . The results contrast with the effect of Cd2+ exposure on radiation lethality, for which sensitization was previously reported in cells from lag and stationary phase cultures. Q J Exp Physiol, 1984 Jan, 69(1), 97 - 115 Effect of the serum concentration of the growth medium on the sodium pump site density of cultured HeLa cells; Aiton JF et al.; The density of sodium pump sites in the plasma membrane of cultured HeLa cells has been measured as a function of the serum concentration of the cell growth medium . Growth in media containing increased concentrations of serum (from 1 to 20% v/v) leads to an increase in sodium pump site numbers (as measured by the specific binding of {3H}ouabain) and pump activity (as measured by the ouabain-sensitive 86Rb influx) . Time-course studies show that (1) new sodium pump sites first appear some 3-6 h after transfer to medium containing an elevated serum concentration and (2) the serum-mediated increase in new sodium pump sites is completely abolished by the protein synthesis inhibitors, cycloheximide and actinomycin D . These results suggest that de novo protein synthesis is required for the development of the serum response . Preliminary characterization of the serum factor responsible for initiating the synthesis of new sodium pump sites indicates that the activity is associated with a high molecular weight serum fraction (greater than 50000) . The different types of interaction seen between the serum effect and other experimental manoeuvres which initiate the synthesis of new pump sites (growth in low-K+ medium, growth in Li+ medium and pre-treatment with exogenous ATP) suggest that there may be more than one pathway for the control of sodium pump site synthesis in cultured HeLa cells. Neoplasma, 1984, 31(4), 385 - 97 Carcinoembryonic antigen, a tumor-associated glycoprotein induces defective lymphocyte function; Hakim AA; Standard carcinoembryonic antigen(s) (CEA) obtained commercially, and CEA preparations from cell membranes and spent media of cultured human malignant melanotic melanoma (HMMC-ShAc and HMMC-ShAm) and of human colon adenocarcinomas (Levo c, Levo m, SW-403 c and SW-403 m) suppressed human peripheral blood lymphocyte (PBL) . CEA preparations obtained from products of in vitro synthesis mediated by mRNA coding for CEA enhanced, whereas glycosylation-deficient preparations of CEA obtained from tumor cells harvested from growth media supplemented with nontoxic levels, i.e . 0.3 microgram/ml of tunicamycin, had no effect on immunoglobulin synthesis by human PBL . CEA caused defective lymphocyte: sheep red blood cell rosette production . It is suggested that CEA is an immunoregulatory glycoprotein synthesized by human tumor cells . The carbohydrate moiety of the glycoprotein is the regulatory determinant. Mol Gen Genet, 1984, 197(2), 309 - 17 Covalent modification of bacterial glutamine synthetase: physiological significance; Kustu S et al.; Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) {(L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2} is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible . These studies further indicated that adenylylated GS is the less active form in vitro . To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S . typhimurium that are unable to modify GS and we have determined the basis for these growth defects . The glnE strains, which lack GS adenylyl transferase activity (ATP: {L-glutamate: ammonia ligase (ADP-forming)} adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH4+) . The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to approximately 10% that under preshift conditions . Consistent with this view, recovery of a rapid growth rate on NH4+ is accompanied by an increase in the glutamate pool . The glnE strains have normal ATP pools after shift . They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth . We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH4+ -excess conditions and thereby to allow rapid growth. Mol Gen Genet, 1984, 195(1-2), 228 - 33 glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation; Castano I et al.; The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac) . Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products . The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF::Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ::Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ . The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome. Arch Virol, 1984, 82(3-4), 241 - 6 Reversible resistance to the antiviral action of interferon observed in altered BHK cells resistant to HVJ (Sendai virus) infection . Brief report; Kimura Y et al.; Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a resistance to the antiviral action of both type I and II interferons . No evidence for a direct inactivation of interferon molecules during incubation of BHK-R cells was obtained . After serial subculture of BHK-R cells in growth medium free of HVJ, surface membranes with the proper sialic acid residues were restored and the cells became susceptible to the interferon action . It is suggested that binding sites for interferons might be ranked above HVJ receptors in the "receptor gradient". Vet Med Nauki, 1984, 21(6), 28 - 33 {Multiplication of Aujeszky's disease virus MK-25 on chick fibroblast cell cultures}; Velichkova M et al.; Attempts were made to produce multilayer cell cultures of chick fibroblasts in a RC-42 roller as well as to replicate in these the Aujezsky's disease virus . Used were fibroblasts of 10-11-day-old chick embryos . The cells were obtained in a closed system after the classic method with an electromagnetic stirrer and a 0.3 percent trypsin in phosphate buffer, having no Ca and Mg ions . Hank's solution was used as a nutrient growth medium, containing 0.5 per cent lactoalbumin hydrolysate and calf serum at pH 7.4-7.6 . Both the cell growth and the cytopathic effect of the virus in the stationary and the roller cultures were carried out simultaneously through microscopy, and the evaluation was made after a fourgrade system . It was found that proper cell cultures or chick fibroblasts could be obtained under the following optimal parameters: volume of nutrient medium--200 cm.3; amount of normal inactivated calf sera--10 per cent; and speed of rotation--10 r . p . m . The titer of the virus was found to surpass by 2 logarithms the virus in the stationary cell cultures for a twice shorter period of cultivation. Mol Gen Genet, 1984, 195(1-2), 29 - 34 Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae . II . The isolation and dosage effect of the regulatory gene GAL80; Nogi Y et al.; The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes . Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene . The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA . When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered . Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium . The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme . From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another. J Membr Biol, 1984, 80(1), 81 - 9 Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells; Nelson DJ et al.; The apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique . We defined the apical membrane as that membrane in contact with the growth medium . In about 50% of the patches, channels with single-unit conductances of 360 +/- 45 pS in symmetrical 105 mM NaCl solutions, and characteristic voltage-dependent inactivation were observed . Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl- over Na+ of about 9:1, calculated from the reversal potential using the constant-field equation . The channel was most active at membrane potentials between +/- 20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity . Reactivation from this inactivation was slow, sometimes requiring minutes . In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels . The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers. J Biol Chem, 1983 Dec 25, 258(24), 15087 - 90 Regulation of sodium-coupled glucose transport by glucose in a cultured epithelium; Moran A et al.; Cultured porcine kidney cells (LLC-PK1) form polarized epithelia that transport glucose from apical to basal surface as in the renal proximal tube . The ability of these cells to transport glucose is known to increase as the epithelium forms and matures in culture . We find that epithelia grown in medium containing 25 mM glucose have reduced hexose transport compared to epithelia grown in 5 mM glucose . This difference is not the result of differences in seeding efficiency and can be reversed by changing the concentration of glucose in the growth medium . Increased transport in epithelia grown in 5 mM glucose is the result of increased influx on the sodium-coupled apical membrane transporter rather than changes in efflux . This difference is apparently the result of more apical membrane transporters in epithelia grown in 5 mM glucose . The number of high affinity phlorizin-binding sites is greater in epithelia grown in 5 mM glucose (about 0.8 pmol/10(6) cells) than in 25 mM glucose (about 0.25 pmol/10(6) cells) . The increase in the number of glucose transporters induced by the low glucose medium is specific in that there is not a comparable change in activity of marker enzymes (alkaline phosphatase, acid phosphatase, or glucose 6-phosphatase) . The nature of the intracellular signal elicited by extracellular glucose remains to be determined. J Biol Chem, 1983 Dec 25, 258(24), 14874 - 9 Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum . Evidence for an unusual acid-stable phosphodiester; Freeze HH et al.; Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts . In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes . {3H}Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion . Approximately 75% of the glycopeptides contained sulfate residues . These could be removed by solvolysis without degrading the underlying oligosaccharide . Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units . Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester . About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species . The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger . Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose . The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters. Pharm Weekbl Sci, 1983 Dec 16, 5(6), 319 - 24 Determination of formation constants of copper(II) complexes of Adler medium components with a solid-state copper(II) ion-selective electrode; Eriks JC et al.; A general method for the determination of overall conditional formation constants of copper(II) complexes of mycoplasma growth medium (Adler) constituents together with the protonation constants of the complexing ligands was developed . In Adler medium a total of 0.204 +/- 0.004 molar copper binding sites proved to be present with log beta 2 = 8.14 +/- 0.05 (mu = 0.11) and log Ka = 6.14 +/- 0.004 . Conditional overall formation constants were calculated at various pH values . Free copper(II) ion concentrations were calculated as a function of the total added amount of copper(II) and pH . The consequences of these findings for the determination of the growth inhibition of mycoplasmas by copper complexes of 2,2'-bipyridyl analogues are stressed. J Parasitol, 1983 Dec, 69(6), 1072 - 8 The effects of 20-hydroxyecdysone and juvenile hormone III on tick cells; Kurtti TJ et al.; Two cell lines isolated from Rhipicephalus appendiculatus ( RAE 25) and Anocentor (= Dermacentor) nitens (ANE 58) responded to the invertebrate hormones 20-hydroxyecdysone (20-HE) and juvenile hormone III (JH III) in vitro . In the presence of 0.2 or 2 nMolar 20-HE, the cells of the continuous line RAE 25 attached to the culture substrate at a rate of 9% per hr for the first 8 hr, as did cells in growth medium . Twenty or 200 nMolar of 20-HE reduced the rate of cell attachment to 6% per hr, and in the higher hormone concentration the cells ceased to attach after 4 hr . Low concentrations (0.2 and 2 nMolar ) of 20-HE stimulated the growth of the RAE 25 line (P less than 0.02), but 200 nMolar or more inhibited growth (P less than 0.001) . Twenty-HE suppressed the growth of the young line ANE 58 in a dose-dependent manner, but the decrease in cell growth was less pronounced than in RAE 25 . Ten to 100 times more (2 and 20 mu Molar) 20-HE was needed to achieve significant growth suppression (P less than 0.025 and less than 0.005) . The growth of both lines declined (P less than 0.01) by 20% ( RAE 25) or 30% (ANE 58) when the medium contained 38 mu Molar of JH III . The bimodal growth response of line RAE 25 to 20-HE also occurred in the presence of 3.8 and 38 mu Molar JH III, and 2 nMolar 20-HE counteracted the suppressive effect of 38 mu Molar JH III.(ABSTRACT TRUNCATED AT 250 WORDS) Antonie Van Leeuwenhoek, 1983 Dec, 49(6), 537 - 49 Respiratory pathways in Hansenula saturnus; Viola AM et al.; Hansenula saturnus is a petite-negative yeast species which displays a different pattern of respiration depending on the age of the cultures . The respiration is sensitive to antimycin A (AA) in the early exponential phase, is sensitive to the simultaneous addition of AA and salicylhydroxamic acid (SHAM) in the middle exponential phase and is sensitive to SHAM in the late exponential and stationary phase . The three respiratory activities are all associated to the mitochondrial fraction . The presence of AA in the growth medium determines the induction of the AA + SHAM-insensitive respiration which is 50% inhibited by 5 mM azide . On the contrary, the presence of erythromycin in the growth medium, which inhibits mitochondrial protein synthesis in this yeast species and the synthesis of cytochromes aa3 and b, totally prevents the appearance of AA + SHAM-insensitive respiration . Moreover, the antibiotic affects cell viability, suggesting a role of the mitochondrial protein synthesis in the cell cycle of H . saturnus. Radiat Res, 1983 Dec, 96(3), 497 - 504 The radiation response of hypoxic cells in EMT6 spheroids in suspension culture does model data from EMT6 tumors; Franko AJ et al.; Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation . These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen . Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors . In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells . The two causes for the oxygen depletion in sealed growth flasks were quantitated . Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase . Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids. J Neurosci, 1983 Dec, 3(12), 2403 - 13 Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment; Schacher S et al.; Neurons from the abdominal ganglion of the mollusc Aplysia californica regenerate neurite processes in dissociated cell culture . Both the nature of neurite outgrowth and the morphology of the cells are influenced by the presence of adult Aplysia hemolymph in the growth medium and the presence of a portion of a cell's original axonal process . Aplysia hemolymph enhances cell survival, the initiation of neurite outgrowth from multiple sites on the cell body surface, the linear growth of the processes, and the amount of branching by those processes . Hemolymph also decreases the diameter of the outgrowing neurite fascicles and the diameter of the individual neurites within the fascicles . The presence of a cell's original axon reduces the time required for the initiation of neurite outgrowth and restricts the formation of multipolar processes . In addition, the presence of an initial axonal segment is essential for neurite regeneration from large adult neurons. J Bacteriol, 1983 Dec, 156(3), 1158 - 64 Incorporation of chlorinated alkanes into fatty acids of hydrocarbon-utilizing mycobacteria; Murphy GL et al.; The cellular fatty acid composition of Mycobacterium vaccae JOB5 and Mycobacterium convolutum R22 was examined after growth on n-alkanes and compared with the fatty acids of the organisms after growth on 1-chlorohexadecane and 1-chlorooctadecane . Growth on n-alkanes resulted in normal fatty acid profiles . Mass spectral analyses indicated that, after growth on the terminally chlorinated n-alkanes, 75 to 86% of the fatty acids in M . convolutum and ca . 55% of the fatty acids in M . vaccae contained chlorine . Neither organism could utilize chloroacetate or 3-chloropropionate as sole source of carbon and energy . When these compounds were added to a growth medium with n-hexadecane as substrate, there was no evidence that chlorinated fatty acids were produced . Terminally chlorinated n-alkanes can be added to the list of n-alkanes, alkenes, and cyclohexylalkane derivatives that can be directly incorporated into cellular fatty acids of hydrocarbon-utilizing organisms. J Natl Cancer Inst, 1983 Dec, 71(6), 1177 - 82 Role of plasminogen in matrix breakdown by neoplastic cells; Bogenmann E et al.; Destruction of the extracellular matrix is often observed during tumor invasion, and proteolytic enzymes may participate actively in the degradation of matrix proteins . The present report elucidates the role of plasminogen in the degradation by tumor cells of an in vitro elaborated extracellular matrix . Matrices produced by rat smooth muscle cells in the presence of {3H}proline or {3H}fucose were used as substrates for human fibrosarcoma cells (HT-1080), mouse melanoma cells (B16F1), or human rhabdomyosarcoma cells (RD) . All three cell lines degraded part of the glycoprotein compartment of the matrix . HT-1080 cells digested the matrices in a density-dependent manner, and while matrix glycoprotein degradation was plasminogen-dependent at the beginning of the experiment and at low cell densities, the zymogen was not essential for further glycoprotein digestion at high cell densities . Depletion of plasminogen from the growth medium resulted in a threefold reduction of matrix degradation by B16F1 cells showing a distinct plasminogen dependency at low cell numbers . RD cells digested only matrix glycoproteins, and this degradation was completely dependent on the presence of plasminogen at all cell densities . These results suggested that plasmin generated from plasminogen by a tumor cell-associated plasminogen activator may be most important for matrix hydrolysis at low cell densities, and while certain tumor cell lines showed a definite plasminogen-independent matrix degradation with increased cell numbers, other neoplastic cells hydrolyzed the matrix only in the presence of the zymogen at all cell densities. Infect Immun, 1983 Dec, 42(3), 1152 - 8 Lymphokine-mediated inhibition of Chlamydia replication in mouse fibroblasts is neutralized by anti-gamma interferon immunoglobulin; Byrne GI et al.; Experiments were carried out to characterize immunologically mediated chlamydial persistence in cell culture . Mouse fibroblasts were activated to restrict Chlamydia psittaci 6BC replication by including mitogen (concanavalin A)-induced spleen cell supernatant fluids from immunized animals in the growth medium . When mouse fibroblasts were incubated with lymphokine for 24 h before infection and then with growth medium after infection (preinfection treatment), chlamydial replication was delayed but eventually detected . No substantial chlamydial growth occurred, even with extended incubation times when mouse fibroblasts were continuously exposed to lymphokine before and after infection . Low levels of infectious chlamydiae were produced in preinfection-treated mouse fibroblasts but not in mouse fibroblasts subjected to continuous lymphokine exposure . Incubation of lymphokine with anti-murine gamma interferon immunoglobulin neutralized the observed lymphokine-mediated activity, but incubation in the presence of anti-murine alpha plus beta interferon serum did not alter lymphokine activity . We conclude that the lymphokine components responsible for activating fibroblasts to restrict C . psittaci replication exhibits properties similar to gamma interferon. J Bacteriol, 1983 Dec, 156(3), 1214 - 21 Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation; Hien NH et al.; The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation . Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V) . The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined . Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells . Glucanase IIIA was the predominant enzyme in stationary resting-phase cells . Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth . Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S . cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes . Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity . Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme . Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium. Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7576 - 80 Developmental regulation of the Aspergillus nidulans trpC gene; Yelton MM et al.; We have cloned the trifunctional trpC gene from Aspergillus nidulans by hybrid phage lambda complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity . Four different phages sharing a 4.3-kilobase region were obtained . Plasmid subclones containing this region also complemented the E . coli trpC mutant . We determined that a 1.8-kilobase DNA fragment was minimally required for complementation . The fragment hybridized with two poly(A)+ RNAs, 3.0 and 3.2 kilobases in length . We infer that these transcripts are Aspergillus trpC mRNAs and that the entire Aspergillus trpC gene is not required for complementation in E . coli . Levels of both trpC transcripts in poly(A)+ RNA are regulated by growth medium composition . They were highest when cells were grown in minimal medium containing nitrate as the nitrogen source and lowest when cells were grown in medium containing yeast extract . The concentrations of the transcripts are also regulated during conidiophore development . Conidiating cultures grown on medium containing yeast extract had significantly higher levels of both transcripts than did hyphae grown in minimal medium containing nitrate . Levels of the transcripts in mature spores were equivalent to those found in hyphae grown in minimal medium containing nitrate . Results from nutritional experiments with an A . nidulans trpC mutant suggest that developmental regulation of trpC mRNA levels may be related to a high requirement for tryptophan or a compound derived from tryptophan during conidiation. Brain Res, 1983 Dec, 313(2), 235 - 44 A quantitative analysis of intramembranous particles during the development of neuroblastoma X glioma hybrid cells; Furuya S et al.; The process of neural differentiation of neuroblastoma X glioma hybrid cells (NG108-15 cells) was studied by freeze-fracture electron microscopy . The distribution of intramembranous particles (IMPs) was random and their density increased in both P-, and E-face of the plasmalemma when the membrane excitability developed . Undifferentiated cells grown in growth medium for 2-6 days had an average of 1300-1500 IMPs/micron2 in the P-face and 250-300/mu 2 in the E-face . After the cells were differentiated with dBcAMP for 1 week, the density of IMPs was 2600/micron2 in the P-face and 540/micron2 in the E-face . The analysis of size distribution showed the increase of the number of medium (7.5-10 nm) and large (greater than 10 nm) particles . The slight increase of large particles was observed in the case of differentiated N18TG2 (the parental neuroblastoma clone) cells . Treatment with dBcAMP did not affect the number and the size distribution of IMPs in the C6BU1 (the parental glioma clone) cells. J Gen Virol, 1983 Dec, 64 ( Pt 12), 2679 - 96 Characterization of eukaryotic transcriptional control signals by assay of herpes simplex virus type 1 thymidine kinase; Lang JC et al.; We describe the characteristics of a general assay for eukaryote transcription-control sequences using the herpes simplex virus (HSV) thymidine kinase (tk) gene . After transfection of cultured cells with tk-containing recombinant plasmids, two assays were used to measure gene expression: short term or transient levels of tk mRNA and TK enzyme activity, and the rate of biochemical transformation from a TK- to a TK+ phenotype in selective growth medium (HAT) . Deletion of the endogenous tk promoter results in 500-fold inactivation of gene expression . Replacement with exogenous transcription-control sequences from the human epsilon globin, mouse beta major globin, simian virus 40 and Moloney murine sarcoma virus (MoMuSV) genomes results in reactivation of gene expression . The presence of enhancers or activators of gene expression can also be conveniently measured . The transient expression assay ranged over two orders of magnitude while the transformation assay was almost two orders of magnitude more sensitive using the same recombinants . Analysis of the transcription-control domains in the MoMuSV LTR sequences shows the presence of both an enhancer and a promoter whose activity equalled that of the tk endogenous promoter . Insertion of the LTR promoter between the LTR enhancer and the tk promoter had little effect on modulating gene expression, suggesting no absolute preference for proximal promoters by this element . The different levels of gene expression obtained appears to be mediated by transcriptional control of full-length tk mRNA . There was an apparent correlation between the results obtained with the transient expression and transformation assays . However, cultured transformed cells all contained roughly the same levels of tk DNA, tk mRNA and tk enzyme activity . We propose that initial expression levels have a major effect in determining the transformation efficiency but that additional genetic controls are superimposed in cells grown in selective HAT medium. J Histochem Cytochem, 1983 Dec, 31(12), 1363 - 6 Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry; Chapman CM et al.; Two components of the cellulase complex (E.C . 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level . Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae . Both enzymes were also present in the culture medium . Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium. J Biol Chem, 1983 Nov 25, 258(22), 13653 - 7 Survival and induction of recA protein in mitomycin C-treated Escherichia coli rec, lex, or uvr strains; Giacomoni PU; The capability to synthesize recA protein has been tested for Escherichia coli treated with mitomycin C . recA protein was assayed using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P . U . (1982) Biochimie 64, 239-246) . Mitomycin C-treated wild type E . coli can express recA gene in a similar quantitative fashion, independently of the growth media used in this work; glucose did not inhibit induction of recA protein in cells growing in synthetic media . Wild type E . coli recovering from energy starvation displays a similar qualitative capability to induce the synthesis of recA protein independently of the stage of growth at which the cells are treated with the drug . At midexponential phase, the cells appear to have an enhanced capability to synthesize recA protein . The relationship between survival and capability to synthesize recA protein was explored for E . coli lex, rec, and/or uvr mutants, after treatment with mitomycin C . A good correlation was found, except for a recB mutant and for an ethidium-sensitive strain, both able to produce as much recA protein as the wild type but 100-fold more sensitive to the drug . A similarly satisfactory correlation was found when plotting the survival after UV irradiation versus the capability of synthetizing recA protein with the exception of an uvrA strain and of a lexA strain. J Biol Chem, 1983 Nov 10, 258(21), 13160 - 5 A biosynthetic role for carnitine in the yeast Torulopsis bovina; Emaus RK et al.; The mode of action of carnitine on the growth of the yeast Torulopsis bovina ATCC 26014 was investigated . When 0.5-5 microM L-carnitine was added to the medium, the growth rate doubled for both aerobic and anaerobic cultures . Cells grown in the absence of carnitine contain 0.4 nmol of L-carnitine/g, wet weight, but with 5 microM L-carnitine in the media, cells contain 1400 nmol of carnitine/g, wet weight, by the end of exponential growth . When {1-14C}acetyl-L-carnitine was added to growth media, almost all of the radioactivity became cell-associated . Most of the 14C was incorporated into cell protein although considerable 14C was recovered in the fatty acid fraction of saponified cells . Analyses of the amino acids derived from radiolabeled protein showed that the acetyl{14C} of acetylcarnitine was in glutamate, arginine, proline, leucine, and lysine . In contrast, {1-14C}acetate labeled leucine and lysine . Isopycnic density gradient analysis demonstrated that carnitine acetyltransferase was primarily associated with mitochondria, while acetyl-CoA synthetase and acetyl-CoA hydrolase were cytosolic . Isolated mitochondria incorporated {14C}acetylcarnitine radioactivity into citrate and 2-oxoglutarate . The data are consistent with carnitine facilitating the transfer of acetyl groups from the cytosol into mitochondria for synthesis of citrate and its metabolites . These results demonstrate a role for carnitine in biosyntheses in the yeast T . bovina. In Vitro, 1983 Nov, 19(11), 853 - 62 Altered sterol synthesis and its relationship to fluid-phase endocytosis in a macrophage cell line P388D1; Miller SC et al.; In a previous study glucocorticoids have been shown to depress the rate of fluid-phase endocytosis in a macrophage cell line, P388D1 . This effect was observed when either fluorescein-labeled dextran or horseradish peroxidase (HRP) was used to measure endocytosis . In this report the relationship between cholesterol synthesis and endocytosis was examined in light of the ability of glucocorticoids to inhibit cholesterol biosynthesis . Two known inhibitors of cholesterol biosynthesis, ML-236B and 25-hydroxycholesterol (25-OH), were compared with dexamethasone (dex) for the ability to suppress endocytosis in cells grown in media supplemented with either 10% whole or delipidized neonatal bovine serum (NBS) . In 10% whole serum all inhibitors reduced the uptake of HRP after 12 h incubation . Dexamethasone (1 microM) suppressed endocytosis by 30% whereas 25-OH (2.5 microM) and ML-236B (11.6 microM) inhibited by 38 and 52%, respectively . Supplementation of the growth medium with mevalonolactone (3.4 mM) prevented the inhibition of endocytosis by ML-236B . In contrast, mevalonolactone supplementation did not prevent either dex or 25-OH from suppressing endocytosis . The same pattern of results was obtained when cultures were grown in delipidized NBS . After 4 h all inhibitors caused a decrease in amount of {14C}acetate incorporated into both nonsaponifiable lipids and digitonin precipitable sterols . Although dex inhibited cholesterol biosynthesis, total cellular cholesterol was unaffected by dex treatment after 24 h incubation . It is suggested that in addition to suppressing mevalonate synthesis, 25-OH, and by analogy dex, may act at some metabolic site(s) distal to the formation of mevalonate. J Bacteriol, 1983 Nov, 156(2), 941 - 4 Sulfite oxidase activity in Thiobacillus novellus; Southerland WM et al.; Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate . Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities . The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate . Tungsten inhibited the growth of T . novellus and the expression of sulfite oxidase activity. Exp Cell Res, 1983 Nov, 149(1), 37 - 44 Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells; Verhoef V et al.; Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine . They do not, however, possess enzymes to form adenine by the cleavage of adenosine . For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase . We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement . A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity . These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements. J Bacteriol, 1983 Nov, 156(2), 975 - 8 Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12; Villarejo M et al.; Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants . This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity . Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression. Proc Natl Acad Sci U S A, 1983 Nov, 80(21), 6581 - 5 Transformation of human skeletal muscle cells by simian virus 40; Miranda AF et al.; Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue . Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives . At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen) . After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities . Transformed cultures also lost contact inhibition of growth and became anchorage independent . Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form . Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA . Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus . These studies demonstrate that human myoblasts can be immortalized by SV40 . This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis. J Biol Chem, 1983 Oct 25, 258(20), 12448 - 54 Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells; Ayusawa D et al.; Thymidylate synthase-negative mutants of cultured mouse cells were immediately committed to cell death upon thymidine deprivation, especially when the cells were synchronized in the S phase . Thymidylate deprivation induced single strand breaks in chromosome-size DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with small fragments, the latter about the size of T4 DNA . An increase in the small DNA fragments paralleled that of thymineless death . Thymidine deprivation also produced double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death . Double-stranded DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above small DNA fragments . Therefore, the strand breaks seemed to occur in some defined portions of the genome and in a specific manner compared to breaks induced by x-rays, which occurred rather randomly . Cycloheximide blocked both thymineless death and the production of the small DNA fragments . The strand breaks induced by thymidine starvation were not repaired but instead advanced on subsequent incubation of the cells in growth medium containing thymidine. Biochim Biophys Acta, 1983 Oct 18, 760(2), 246 - 55 Antibacterial effect of the scandium complex of enterochelin . Studies of the mechanism of action; Plaha DS et al.; There is good evidence to show that ferric enterochelin is an essential growth factor for a number of Gram-negative pathogenic bacteria exposed to the host iron binding proteins, transferrin and lactoferrin . Tests of nineteen complexes of enterochelin as potential antibacterial agents showed that only those containing either indium (In3+) or scandium (Sc3+) inhibited bacterial growth . In this study, further evidence is presented which demonstrates a competition between the Sc3+ and Fe3+ complexes . The uptake of both complexes is energy dependent and is also repressed in iron-replete cells . The Sc3+ complex accumulates within the cells at 20% of the rate of the Fe3+ complex . The main components of the ferric enterochelin transport system are required for the transport of the Sc3+ complex although some Sc3+ appears to enter the cell by another route . The accumulation, within the cell, of 14C-labelled enterochelin complexes depends on the growth medium . The relationship of the size of the metal ion to the biological activity of the complex is discussed and possible mechanisms of action of the Sc3+ complex are considered. Biochem J, 1983 Oct 15, 216(1), 37 - 42 Biosynthesis of intestinal microvillar proteins . Role of the Golgi complex and microtubules; Danielsen EM et al.; The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestinal explants . On the ultrastructural level, monensin (1 microM) caused an increasingly extensive dilation and vacuolization of the Golgi complex during 4h exposure of the explants . On the molecular level, the effect of monensin was twofold . (1) The processing from the initial high-mannose-glycosylated form to the mature complex-glycosylated form was arrested . For some of the enzymes studied, intermediate stages between the high-mannose and complex forms could be seen, probably corresponding to 'trimmed' or partially complex-glycosylated polypeptides . (2) Labelled microvillar enzymes failed to reach their final destination . These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes . The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes in the microvillar membrane during a 3 h labelling period . Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the microvillar membrane. Biochim Biophys Acta, 1983 Oct 13, 741(1), 1 - 6 Thiolated nucleotides in yeast transfer RNA; Laten HM et al.; By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast . We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S . cerevisiae tRNA . In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification. Sex Transm Dis, 1983 Oct-Dec, 10(4 Suppl), 244 - 6 Biochemistry of Mycoplasma hominis; Vinther O; The growth of Mycoplasma hominis is stimulated by arginine . A possible mechanism for degradation of this amino acid is the arginine dihydrolase pathway . The first enzyme of the pathway, arginine deiminase, is inducible in M . hominis . Evidence exists that the dihydrolase pathway is not the major pathway to production of adenosine triphosphate in the organism . M . hominis does not take up nucleosides from the growth medium, although the metabolic processes required for the transformation of precursors into nucleic acids are present . The system by which L-methionine is transported across the cellular membrane of M . hominis resembles the active-transport systems found in other microorganisms . The membrane of M . hominis contains a high level of cholesterol in the unesterified form . The low-density fraction of human serum lipoprotein is an effective cholesterol donor, as are cholesterol-phospholipid liposomes with a high cholesterol content. Can J Microbiol, 1983 Oct, 29(10), 1350 - 60 Acid and alkaline phosphatases of Capnocytophaga species . I . Production and cytological localization of the enzymes; Poirier TP et al.; The two hydrolytic enzymes, acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase, of the three types species of Capnocytophaga were examined . Both enzymes were produced constitutively, with their activity highest in C . ochracea strain 25 . These two degradative enzymes (approximately 10% of the total activity) were released into the growth medium during the latter stages of growth, both as soluble and membrane-bound enzymes . When grown in the presence of high concentrations of organic phosphates, the synthesis and expression of AcP and AlP was unaltered . Cyto- and immuno-chemical localization situated the phosphatases in the periplasmic space, at the cell surface, and in membranous vesicles. Biochem J, 1983 Oct 1, 215(1), 107 - 16 Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels; Gallagher JT et al.; Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium . Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium . Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels . In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface . The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate . The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions. J Cell Physiol, 1983 Oct, 117(1), 109 - 15 Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture; Kano-Sueoka T et al.; Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture . The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn . In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not . Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out . In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased . The increase in PE content leveled off at 10 microM . Further, the increase in PE content was correlated with increased rate of growth . In contrast, PE content or growth rate did not change at all in 22-1 cells . In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells . De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells. J Bacteriol, 1983 Oct, 156(1), 369 - 74 Metabolic activities of isolated akinetes of the cyanobacterium Nostoc spongiaeforme; Thiel T et al.; Intact akinetes (spores) of the cyanobacterium Nostoc spongiaeforme can be isolated free of vegetative cells and heterocysts . The akinetes remain viable for at least 2 weeks in distilled water . They do not germinate in water but do so readily when transferred subsequently to cyanobacterial growth medium . Isolated, nongerminating akinetes incorporated 35S from Na235SO4 into protein and lipid . Similar incorporation was observed when akinetes were isolated from old cultures (containing primarily akinetes) which were labeled with Na235SO4 for 4 to 5 h before isolation . The metabolic activities of isolated akinetes were therefore not a factitious response to the isolation procedure . Autoradiographs of radioactive akinetes showed that 35S was incorporated by virtually all akinetes, rather than by a small subpopulation of active cells . Akinetes consumed O2 in the dark and, in a dichlorophenyl dimethylurea-sensitive reaction, evolved O2 in the light . We conclude that akinetes are metabolically active under conditions in which germination does not occur. J Inorg Biochem, 1983 Oct, 19(2), 105 - 17 The relationship of chromium to the glucose tolerance factor . II; Haylock SJ et al.; After incubation with CrCl3 X 6H2O (or 51CrCl3 X 6H2O) for 25 days, a sterile growth medium, whole yeast cells harvested after growth on a similar chromium-containing medium for the same period, and the spent growth medium remaining after removal of the yeast were each subjected to the separation procedure reported previously {S . J . Haylock . P . D . Buckley and L . F . Blackwell, J . Inorg . Biochem., in press} . The results obtained showed that most of the eleven chromium-containing fractions isolated previously were artifacts formed as a result of direct reaction between the chromium and components of the medium . An anionic complex (which was the major chromium-containing fraction isolated) was identified as a chromium-glucose complex, but one possessing no biological activity . The biologically active chromium-containing fractions (P-3 and P-4) that were only present after yeast had been grown in the medium were further purified, however, during the purification steps, the biological activity was cleanly separated from the chromium material for both P-3 and P-4 . Fraction P-4 was subsequently shown to consist of approximately 90% tyramine, but pure tyramine was not active in the yeast bioassay . Although the structure of the glucose tolerance factor-active component in fraction P-3 could not be determined due to the presence of high concentrations of salt that could not be separated on gel filtration columns, the results show that the glucose tolerance factor from brewer's yeast can no longer be regarded as a chromium complex. J Bacteriol, 1983 Oct, 156(1), 136 - 40 Cleavage and resynthesis of peptide cross bridges in Escherichia coli murein; Goodell EW et al.; In Escherichia coli, peptide cross bridges in the murein undergo turnover after they are synthesized . Peptide cross bridges formed in the presence of {3H}diaminopimelic acid were found to lose 3H label from their donor peptides after the {3H}diaminopimelic acid was removed from the growth medium . There was a corresponding increase in the amount of 3H label in acceptor peptides so that the total amount of label in the peptide cross bridges remained constant . Our explanation of this observation is that the cross bridges are cleaved by the cell, and the original 3H-labeled donor peptides are incorporated into new cross bridges . Since these 3H-labeled peptides are now only tetrapeptides, they can only be used as acceptors when new cross bridges are formed. Exp Cell Res, 1983 Oct, 148(1), 95 - 103 Fusion-mediated implantation of band 3 into living cells . A new system to study degradation of membrane proteins; Beigel M et al.; Sendai virus (SV) glycoproteins were co-reconstituted with the erythrocyte membrane polypeptide designated as band 3 (B3) . Fusion of the hybrid vesicles, SV-B3, with L, HeLa and Friend erythroleukemic (FEL) cells led to fusion-mediated implantation of the erythrocyte band 3 into the plasma membranes of the above cells . By the use of radiolabelled band 3 {( 3H}H2DIDS-B3) it was possible to demonstrate that about 1-4 X 10(6) molecules of band 3 can be transferred to the membranes of each of the above cell lines . Cultivation of the recipient cells in growth medium resulted in gradual decrease of the cell-associated counts . The reduction pattern followed first-order kinetics, reaching t 1/2 of disappearance at about 25-30 h in the case of L and HeLa cells, and 57 h in the case of FEL cells . NH4Cl added to the growth medium significantly inhibited the disappearance of band 3 during growth . The studies of the present work indicate that the reduction in the cell-associated band 3 during growth may represent degradation of this polypeptide by the recipient cells. Mutat Res, 1983 Oct, 113(6), 499 - 506 A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate; Raveh D et al.; 6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10(3)-10(4) cells/0.2 ml growth medium/0.4 cm2) when selected with 30 microM 6-thioguanine and 0.1 microgram/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells . Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies . After treatment of the V79 cells with the carcinogen benzo{a}pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes . The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure . The active carcinogen benzo{a}pyrene at 1 microgram/ml yielded about 100 mutants per 10(5) colony-forming cells . The same dose of a less active carcinogen, cyclopenta{c,d}pyrene, yielded about 20 mutants per 10(5) colony-forming cells, and benz{a}anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested . Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells. J Cell Physiol, 1983 Oct, 117(1), 9 - 14 Reduced free-methionine in methionine-dependent SV40-transformed human fibroblasts synthesizing apparently normal amounts of methionine; Stern PH et al.; Many different types of cancer cells have been shown to be methionine-dependent . These cells, unlike normal cells, grow poorly or not at all when methionine is replaced by its immediate precursor homocysteine in the growth medium (Met- Hcy+ medium) . We have previously shown that apparently normal total amounts of methionine are synthesized by methionine-dependent SV40-transformed human fibroblasts . However, methionine-dependent cells in Met- Hcy+ medium accumulate reduced amounts of S-adenosylmethionine (AdoMet) and elevated amounts of S-adenosylhomocysteine (AdoHcy) that together probably limit growth . In this report, we demonstrate that the amount of free methionine is low in methionine-dependent SV40-transformed human fibroblasts in Met- Hcy+ medium compared to normal human diploid fibroblasts . In contrast, in Met+ Hcy- medium, the amount of free methionine is comparable in both cell types . The deficient pool of free methionine in methionine-dependent cells in Met- Hcy+ medium allows only low amounts of AdoMet to be formed . However, large amounts of the biosynthesized methionine are channeled into protein synthesis . Possible mechanisms are discussed to explain this cancer-associated metabolic defect. J Cell Physiol, 1983 Oct, 117(1), 51 - 61 Growth stimulatory precipitates of Ca2+ and pyrophosphate; Bowen-Pope DF et al.; Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM . This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H-uridine, and 3H-choline uptake, and the incorporation of 3H-leucine and 3H-thymidine into trichloroacetic acid (TCA)-insoluble material . Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism . The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi . Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations), PPi (77% of anions), and Pi (23% of anions) . Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi . We have distinguished cell surface-bound PPi from intracellular PPi by differential extraction . The quantity of surface-bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation . If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate-containing medium, the cells are not stimulated . These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi . PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I-platelet-derived growth factor to its specific cell-surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen-receptor systems . In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation . The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters. Endocrinology, 1983 Oct, 113(4), 1191 - 6 Ectopic growth hormone-releasing factor and dibutyryl cyclic adenosine monophosphate-stimulated growth hormone release in vitro: effects of corticosterone and estradiol; Webb CB et al.; Glucocorticoids and estrogens each affect GH secretion in vivo . The effects of corticosterone and estradiol (E2) were studied singly and in combination on GH secretion and cell content of primary cultures of rat adenohypophyseal cells grown in media containing intact or hormone-deficient serum . Secretion was measured under basal conditions and in response to maximally stimulatory doses of ectopic GH-releasing factor (E-GHRF) derived from a carcinoid tumor and dibutyryl cAMP {(DBcAMP) 10(-3) M} . Basal GH release measured over a 4-h period was suppressed by 40% (P less than 0.001) when hormone-deficient serum was substituted for normal serum in the growth media, and the stimulatory responses to DBcAMP and E-GHRF were markedly attenuated (P less than 0.001) . The GH content of unstimulated cells was also decreased {29 +/- (SE) 7%, P less than 0.001} . The addition of corticosterone, 3 X 10(-8) M to 3 X 10(-6) M, to the 4-day growth media resulted in dose-related increases in basal and DBcAMP-stimulated GH release during the 4-h test period which was proportional to the increases in total cellular GH content . In contrast, corticosterone exposure caused a dose-related enhancement of E-GHRF-stimulated release above that accounted for by the increase in total GH content alone . Concomitant exposure to the releasing stimuli and corticosterone during a 4-h incubation, however, reduced the effects of the releasing stimuli . The addition of E2, 10(-10) M to 10(-8) M, during the 4-day growth period and/or the 4-h stimulation period did not affect the secretion of GH either basally or in response to the stimuli . E2 did increase the cell content of GH, but the effects were not additive to those of corticosterone . These studies indicate that long term (4-day) exposure to corticosterone increases net GH synthesis and E-GHRF-stimulated release, but that acute (4 h) exposure inhibits stimulated release . Although E2 also increases cellular content of GH, it exhibits no demonstrable direct effects on GH secretion or content in the presence of corticosterone. J Parasitol, 1983 Oct, 69(5), 846 - 9 Pyrimidine metabolism in Tritrichomonas foetus; Jarroll EL et al.; The pyrimidine metabolism of Tritrichomonas foetus (KV 1) was studied using whole cells and cell homogenates . Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids . Orotate and aspartate were not incorporated into pyrimidine bases . Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and uridine kinase) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates . The evidence presented supports the proposal that T . foetus is incapable of synthesizing pyrimidines de novo but is capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and that enzymes of this parasite's pyrimidine salvage pathway are not organelle-associated. Food Chem Toxicol, 1983 Oct, 21(5), 551 - 6 Mutagenic activity of a nitrosated early Maillard product: DNA synthesis (DNA repair) induced in HeLa S3 carcinoma cells by nitrosated 1-(N-L-tryptophan)-1-deoxy-D-fructose; Lynch SC et al.; HeLa S3 cells in suspension were incubated at 37 degrees C with various concentrations of the Amadori compound 1-(N-L-tryptophan)-1-deoxy-D-fructose (Trp-Fru), of its nitrosated analogue NO-Trp-Fru and of sodium nitrite, for varying periods of time, and were assayed for viability (trypan blue exclusion test) and for intracellular DNA, RNA and protein synthesis . None of the compounds tested had any effect on cell viability, or on RNA and protein synthesis apart perhaps from a slightly inhibitory action . While Trp-Fru remained ineffective also as far as intracellular DNA synthesis was concerned, both NO-Trp-Fru and NaNO2 had a major effect on DNA synthesis . With NaNO2, stimulation of DNA synthesis occurred at concentrations above 1 mM in the growth medium, but with NO-Trp-Fru synthesis increased at concentrations below 1 microM . The excess DNA synthesis (i.e . synthesis above control activity) observed with NO-Trp-Fru and also with NaNO2 was due to DNA repair . This was verified by keeping the cells under conditions that prevented normal semi-conservative replication but permitted DNA repair ('unscheduled DNA synthesis') . Two major routes are suggested by which NO-Trp-Fru could damage DNA. In Vitro, 1983 Oct, 19(10), 749 - 58 A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells; Malan-Shibley L et al.; Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin . Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free "attachment medium" was first devised in which cells would attach to the substratum without loss of viability . Then a growth medium that would support cell proliferation was developed . Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response . Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium . Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology . Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. Br J Cancer, 1983 Oct, 48(4), 485 - 93 Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells; Higgins PJ et al.; Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics . The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used . Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent . Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO . The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells . Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation . Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population) . The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion . DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle. Toxicol Appl Pharmacol, 1983 Sep 30, 70(3), 390 - 401 Bone marrow toxicity induced by oral benzo{a}pyrene: protection resides at the level of the intestine and liver; Legraverend C et al.; The Ah locus encodes a cytosolic receptor that regulates the induction of certain drug-metabolizing enzymes by polycyclic aromatic hydrocarbons such as benzo{a}pyrene . Some inbred mouse strains such as C57BL/6N have the high-affinity Ah receptor (Ahb/Ahb), others such as DBA/2N, the poor-affinity receptor (Ahd/Ahd) . Presence of the high-affinity receptor leads to greater cytochrome P1-450 induction by benzo{a}pyrene; in turn, enhanced benzo{a}pyrene metabolism can result in more toxic intermediates or greater detoxication, depending upon the test system studied . Benzo{a}pyrene in the growth medium, in direct contact with cultured myeloid cells, is more toxic to C57BL/6N than DBA/2N cultured cells . Oral benzo{a}pyrene induces P1-450 (measured by benzo{a}pyrene trans-7,8-dihydrodiol formation determined by high-performance liquid chromatography) in C57BL/6N but not DBA/2N intestine and liver . In the bone marrow of oral benzo{a}pyrene-treated C57BL/6N and DBA/2N mice, the magnitude of P1-450 induction is about the same . WB/ReJ (Ahd/Ahd), C57BL/6J (Ahb/Ahb), or (WB/ReJ)(C57BL/6J)F1 (Ahb/Ahd) marrow was transplanted into lethally irradiated (WB/ReJ)(C57BL/6J)F1 mice . DBA/2J (Ahd/Ahd) marrow was transplanted into lethally irradiated BALB/cByJ (Ahb/Ahb) mice and vice versa . Mice having the Ahd/Ahd intestine and liver died in less than 3 weeks of benzo{a}pyrene feeding (120 mg/kg/day), irrespective of the source of transfused marrow . All the data are consistent with pharmacokinetic differences in the tissue distribution of benzo{a}pyrene: mice having the high-affinity receptor, and therefore the P1-450 induction process in the intestine and liver, are protected from oral benzo{a}pyrene-induced myelotoxicity. J Biol Chem, 1983 Sep 25, 258(18), 10997 - 1003 Measurements of S-adenosylmethionine and L-homocysteine metabolism in cultured human lymphoid cells; German DC et al.; The intracellular content and turnover of S-adenosyl-L-methionine (AdoMet) were measured in cultured human lymphoid cells . AdoMet levels were found to be 59 nmol/ml cell volume in exponentially growing WI-L2 lymphoblasts, 3.3-8.1 nmol/ml cell volume in unstimulated peripheral blood mononuclear cells, and 25-33 nmol/ml cell volume 24-48 h after the latter were stimulated with phytohemagglutinin . Increases in the AdoMet content of stimulated cells occurred within 2 h after addition of lectin . First order, pool turnover rates were of the same order of magnitude (0.029-0.091/min) for all three types of cultured cells, but owing to the differences in AdoMet content, absolute utilization rates differed markedly and were 4.5, 0.12-0.40, and 1.41-1.57 nmol/min/ml cell volume in WI-L2, unstimulated peripheral mononuclear cells, and lectin-stimulated peripheral mononuclear cells, respectively . Measurements of homocysteine accumulation in growth medium and of transsulfuration to cysteine indicate that a minimum of 82% of the AdoMet synthesized by WI-L2 is used for transmethylation . Remethylation of homocysteine by these cells could not be detected . AdoMet synthesis accounts for 20-23% of methionine utilization by WI-L2 . Judging from the accumulation of homocysteine in the medium of phytohemagglutinin-stimulated peripheral mononuclear cells, a minimum of 38% of AdoMet synthesized must be used for transmethylation . Even though AdoMet utilization by unstimulated peripheral mononuclear cells is relatively small compared to that of stimulated cells and WI-L2, our data indicate that AdoMet turnover in such "resting" cells is three to five times that estimated for nonhepatic tissues . These findings may be relevant to the hypothesis that lymphoid cells are unusually sensitive to inhibition of transmethylation reactions. Biochim Biophys Acta, 1983 Sep 22, 763(2), 183 - 90 Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture; Hampson IN et al.; Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with D-{1-3H}glucosamine and sodium {35S}sulphate . Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet . Sulphated glycosaminoglycans co-chromatographed when eluted by NaCl gradient from DEAE-cellulose . The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate . Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid . Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units . Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain . The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1 . Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of 'O'-sulphate groups differed strikingly between heparan sulphates from the medium and cell-surface compartments . A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species . The absence of detectable amounts of 35{S}sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups. J Biol Chem, 1983 Sep 10, 258(17), 10378 - 83 Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells; Karenlampi SO et al.; Hepa-1c1c7, a mouse hepatoma cell line, was used to study the effect of cytochrome P1-450 inducers on the binding of 125I-epidermal growth factor (EGF), 125I-insulin, or {20-3H}phorbol 12,13-dibutyrate each to its specific cell-surface receptor . After a 24-h exposure to the cultured cells, several polycyclic hydrocarbon P1-450 inducers decrease the binding of EGF to EGF receptors much more than phenobarbital does . There appears to be a selectivity in the inhibitory effects: whereas EGF binding to EGF receptors is blocked, the binding of either phorbol ester or insulin each to its specific cell-surface receptors remains unaffected . The rank order of binding affinities of these chemicals to the cytosolic Ah receptor (2,3,7,8-tetrachlorodibenzo-p-dioxin much greater than benzo{a}pyrene greater than benzo{a}anthracene greater than 6-aminochrysene much greater than phenobarbital) is not correlated with their effects on EGF binding capacity . The effect of polycyclic hydrocarbons on EGF binding takes 24 h at 37 degrees C to be maximal, whereas phorbol 12-myristate 13-acetate, a potent tumor-promoting compound, inhibits EGF binding in less than 30 min . Removal of benzo{a}anthracene from the growth medium after 24 h results in a gradual recovery in EGF binding, indicating that the effect is reversible . Benzo{a}pyrene and benzo{a}anthracene are relatively ineffective at decreasing EGF binding to the EGF receptors in Hepa-1 mutant clones c2 and c4, which lack a normally functioning Ah receptor and inducible aryl hydrocarbon hydroxylase activity (P1-450) . The very toxic metabolite (+)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene, when added directly to the growth medium of c4 cells, however, is effective at decreasing EGF binding . These data suggest that electrophilic metabolites of polycyclic aromatic compounds, formed by P1-450 induced during the exposure of Hepa-1 cells to these chemicals, are important in decreasing EGF binding to the EGF cell-surface receptor . Occupancy of the Ah receptor per se does not affect EGF binding. J Chromatogr, 1983 Sep 9, 276(2), 279 - 88 Studies of metabolic changes in cell cultures infected with four serotypes of dengue fever viruses by frequency-pulsed electron-capture gas-liquid chromatography; Brooks JB et al.; Monkey kidney cell cultures were infected with four serotypes of dengue viruses, and the supernatant fluids of the cell cultures were extracted for amines, alcohols, carboxylic acids, and hydroxy acids . The derivatized extracts were then analyzed by frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC) . FPEC-GLC profiles of the hydroxy acids showed peaks that were different for different serotypes and the FPEC-GLC carboxylic acid profiles differed from the control medium . These differences were reproducible when the same lot for medium was used . There were differences in profiles between lots of control media due apparently to different fetal bovine sera used in the growth medium . Therefore, the same lot of medium was necessary to reproduce profiles . The data obtained from the study indicate that FPEC-GLC can be used to detect changes in cellular metabolism caused by viral infection, and that these metabolic changes might be useful in detection of genetic differences in viruses as reflected by detectable changes in the metabolism of the infected cell. Lab Invest, 1983 Sep, 49(3), 346 - 52 Altered protein glycosylation and procollagen to collagen conversion in human fibroblasts; Duksin D et al.; Skin fibroblasts, from a 9-year-old girl with an apparent heritable disorder of connective tissue, were cultured in vitro . The biosynthesis of the extracellular matrix glycoproteins, procollagens, and fibronectin was studied using radioactively labeled sugars and amino acids . Glycoproteins synthesized and secreted into the growth medium were found to be only partially glycosylated and the extracellular limited proteolytic conversion of procollagen type I to collagen was impaired, with procollagen chains accumulating in the growth medium . These biosynthetic alterations have not been previously reported in human skin fibroblasts, but similar findings have been described in tunicamycin-treated chick fibroblasts. Onderstepoort J Vet Res, 1983 Sep, 50(3), 149 - 55 Biochemical studies on a lysosomal storage disease in Abyssinian cats; Lange AL et al.; Blood lipid analysis was performed on the serum of 2 normal kittens and 1 adult cat and on serum from 3 affected kittens . Thin layer chromatography was done on tissue extracts of various organs from clinically affected kittens and unaffected unrelated kittens of a similar age, and on serum from carrier cats, affected kittens, related unaffected kittens and unrelated kittens . Spleen and lymph node cell cultures were prepared from 1 affected kitten and the growth medium and cell cultures were analysed for lipids . A lecithin-like phospholipid was identified in the serum of an affected kitten, a carrier cat and a related unaffected kitten . This substance was produced by the liver of affected kittens and also by macrophage-like cells in spleen cell cultures prepared from the spleen of a kitten with signs of the disease. Genetika, 1983 Sep, 19(9), 1426 - 32 {Expression of the amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus . III . The effect of mutations in the rpoB gene coding for the beta-subunit of RNA-poly-merase on ilv-operon expression}; Gordeev VK et al.; The influence of rifampicin resistance mutations on expression of the ilv operon of Escherichia coli was investigated . Some of these mutations, like those previously described in our works, occur in translation machinery of E . coli and inhibit derepression of the ilv operon . However, another group of mutations stimulated the expression of the operon when mutated cells were transferred from rich to minimal growth medium . The possible mechanisms of the effects of rifampicin resistance mutations on the action of coupled transcription--translation system are discussed. Infect Immun, 1983 Sep, 41(3), 1245 - 51 Colonial opacity variation in Mycoplasma pulmonis; Liss A et al.; Colonial size and opacity variation were observed in four independently isolated strains of the murine pathogen Mycoplasma pulmonis . Selecting colonial opacity variants of similar size, we identified opaque and transparent stable variants . Opaque colony-derived broth cultures shed transparent colonies at a rate of about 1.2 X 10(-8) per CFU per generation . The reverse conversion was about two orders of magnitude less frequent . Appearance of opacity and plating efficiency of each pure culture were altered by changing the serum source used to supplement the growth medium . Horse or sheep serum was most efficient at accentuating visualization of opacity differences . Fetal bovine serum was least efficient . In two M . pulmonis strains, each opacity variant showed a distinctive polypeptide profile, as displayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In the same strains, distinctive intrastrain differences were found by agarose gel electrophoresis to display the DNA fragments produced after digestion by several endonucleases . Each pure culture variant retained these differences in DNA even when grown in a medium supplemented with a serum which did not accentuate visualization of the opacity phenotype . Characterization of variants in 30 other M . pulmonis strains is in progress. Eur J Biochem, 1983 Sep 1, 135(1), 33 - 9 Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum-free medium; Broad TE et al.; Fibroblasts from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB 202 supplemented with 1 microgram/ml bovine insulin, 1 microM dexamethasone, 100 ng/ml fibroblast growth factor and 20 micrograms/ml of the lipid preparation described by Bettger, W . J., Boyce, S . T., Walthall, B . J . and Ham, R . G . {(1981) Proc . Natl Acad . Sci, USA, 78, 5588-5592} . When maintained as a confluent monolayer in this medium, the fibroblasts from the adipose tissues spontaneously underwent an adipose differentiation . This was accelerated by substituting medium F12 for medium MCDB 202, and by raising the CO2 tension from 2% to 7.5% in air over the cultures . The differentiation was inhibited by deleting FGF from the growth medium, or by coating the culture surface with fibronectin or poly-D-lysine . Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum . The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of {14C}acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol-3-phosphate dehydrogenase. J Bacteriol, 1983 Sep, 155(3), 1162 - 70 Physiological characterization of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis; Little R et al.; We have previously reported the isolation of Escherichia coli rpoB mutants in which the control of ribosome synthesis by the nucleotide effector guanosine tetraphosphate (ppGpp) is altered, owing to a 20-fold increased sensitivity of the mutant RNA polymerases to ppGpp . In these mutants, the level of ppGpp during exponential growth is decreased about 10-fold, relative to that of rpoB+ wild-type strains, such that a near normal partitioning of RNA polymerase occurs with respect to stable RNA (rRNA and tRNA) gene activity . Here, the physiological effects of two different rpoB alleles in a relA+ and relA background were analyzed in greater detail by comparison with their isogenic rpoB+ wild-type parents . For a given growth medium, the rpoB mutations were found to affect four parameters which resulted in a reduction of growth rate . The results reinforce a previous conclusion that a key element in control of the bacterial growth rate is a mutual relationship between control of ribosome synthesis by ppGpp and control of relA-independent ppGpp metabolism by the concentration and function of ribosomes. Cell Tissue Kinet, 1983 Sep, 16(5), 493 - 504 Epidermal keratinocytes actively maintain their intracellular polyamine levels; Roseeuw DI et al.; Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation . However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed . The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels . The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations . At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine . Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine . Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium . At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium . We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium . Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function. Biochem Biophys Res Commun, 1983 Aug 12, 114(3), 950 - 4 Inhibition of ornithine decarboxylase in human fibroblast cells by type I and type II interferons; Sekar V et al.; Dilution of human fibroblast GM2767 cell cultures into fresh serum-containing growth medium induces ornithine decarboxylase activity 45-fold over a six-hour interval . When the fibroblast cultures are supplemented with human fibroblast alpha-, beta-, or gamma-interferon at the time of dilution into fresh growth medium, the induction of ornithine decarboxylase is inhibited 61%, 90%, and 65%, respectively . beta-Interferon is the most effective type of interferon to inhibit induction of ornithine decarboxylase. J Bacteriol, 1983 Aug, 155(2), 802 - 5 DNases of Acholeplasma spp; Roganti FS et al.; One strain from each of seven species of Acholeplasma was examined for the presence of DNases . Six of the strains were found to be DNase positive when assayed with DNA-methyl-green-containing agar, indicating that this method can be used to differentiate the seventh strain, Acholeplasma axanthum, from the remaining Acholeplasma spp . Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA revealed DNases in the cell extracts of all seven strains and in the supernatant growth medium of five of the strains . The electrophoretic patterns were highly characteristic for each strain and can be used for the rapid identification of different strains of Acholeplasma. J Cell Physiol, 1983 Aug, 116(2), 227 - 35 An estradiol-responsive mouse endometrial cell strain with inducible aryl hydrocarbon hydroxylase activity; Iannaccone PM et al.; A mouse endometrial cell population has been isolated by mild tryptic digestion of the uterine lining . The cells were morphologically similar to endometrial gland cells in the intact mouse endometrial gland . The endometrial cells had a modal chromosome number of 66 . The cells were adherent to glass as well as plastic and contained numerous large refractile, osmophilic, non-membrane-limited granules which stain with periodic acid-Schiff reagent but do not stain with oil red O, Sudan black, or Alcian blue . Cell growth was responsive to 17 beta-estradiol; cell number increased 1.34-fold in 4 days in the presence of 10(-8) M estradiol . The cells are not tumorigenic . The cells showed induction of aryl hydrocarbon hydroxylase (AHH) activity when 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to the growth media for 24 h . AHH activity and its induction were investigated with cells grown in the presence and absence 10(-8) M estradiol . Cells grown in media containing estradiol exhibited a 6.2-fold induction by TCDD; cells grown without estradiol gave an 8.4-fold induction of AHH activity . AHH activity and its induction by TCDD were demonstrated in cells grown with fetal calf serum that had been pretreated with dextran-coated charcoal to remove endogenous steroids . Benzanthracene failed to induce AHH activity significantly. J Protozool, 1983 Aug, 30(3), 606 - 8 Defined minimal growth medium for Acanthamoeba polyphaga; Ingalls CS et al.; Nutritional requirements of Acanthamoeba polyphaga (strain PD) were compared to those reported for A . castellanii . Although A . polyphaga and A . castellanii have essentially the same minimal amino acid requirements--arginine, methionine, leucine, isoleucine, and valine--A . polyphaga cannot utilize acetate as sole carbon source, but A . castellanii can if the medium is supplemented with glycine. J Parasitol, 1983 Aug, 69(4), 633 - 6 High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium; Iovannisci DM et al.; A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar . Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum . Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum . Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency . Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%. J Nutr Sci Vitaminol (Tokyo), 1983 Aug, 29(4), 389 - 98 Precursor of carbon atom five and hydroxymethyl carbon atom of the pyrimidine moiety of thiamin in Escherichia coli; Yamada K et al.; The pyrimidine moiety of thiamin can be synthesized in bacteria from 5-aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis . To transform the imidazole ring of AIR to the pyrimidine, the bond between C-4 and C-5 of the imidazole ring should be ruptured and a two-carbon unit should be inserted . We investigated a precursor of the two-carbon unit which should be inserted to form the pyrimidine . To examine the possibility of the two-carbon compound as a precursor which is formed from a three-carbon intermediate in the glycolytic pathway and its three-carbon derivative by decarboxylation in cells, we studied the incorporation of {2-14C}glycerol, {2-14C}pyruvate, {U-14C}alanine and {3-14C}serine into the pyrimidine by Escherichia coli . Glycerol, pyruvate and alanine were not incorporated into the pyrimidine . Radioactive carbon of {3-14C}serine was incorporated into C-2 of the pyrimidine via one-carbon units pool . Then, we investigated the possibility of a ribose moiety of AIR being a precursor of the two-carbon unit . As ribose has low permeability into E . coli cells, the ribose moiety of AIR was labeled with {14C}glucose which was added to the growth medium . The results show that two radioactive carbons of {U-14C}glucose were incorporated into the pyrimidine and radioactive carbon of {6-14C}glucose was incorporated into hydroxymethyl carbon attached to C-5 of the pyrimidine . The dilution rates of the specific radioactivity of the pyrimidine from {U-14C}glucose and {6-14C}glucose well coincide with those of the ribose moiety of the nucleotide (AMP) . Radioactive carbon of {1-14C}glucose was not incorporated into the pyrimidine and nucleotide . It is concluded that the two-carbon fragment derived from C-4' and C-5' of the ribose moiety of AIR can be incorporated into the pyrimidine moiety of thiamin as the precursor of C-5 and hydroxymethyl carbon. Arch Microbiol, 1983 Aug, 135(1), 12 - 5 On characterization of the growth of Escherichia coli in batch culture; Kahru A et al.; Several growth monitoring parameters, including adenine nucleotide contents, were measured during Escherichia coli K12 batch cultivation in mineral medium with glucose . The adenylate energy charge with its mean value of 0.83 remained roughly stable during growth . The total adenylate and ATP pools (nmol/mg dry weight), and also the individual cell volume changed with a pattern of two maxima at approximately 4 and 10 h of cultivation . After the exhaustion of the glucose from the growth medium the adenylate energy charge, the pools of ATP and total adenylate started to decrease marking the onset of the stationary growth phase . Our data indicate that actually there was only a limited period within the logarithmic growth phase during which the growth might have been balanced: during this period of 1.5 h different growth monitoring parameters (optical absorbance, cell number, total cell volume, and ATP content per ml) increased with almost equal rates . Moreover, as ATP pool and median cell volume during this short period were approximately constant, the culture might have been even in the steady state. Gene, 1983 Aug, 23(2), 105 - 15 The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli; Skogman G et al.; The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media . It was found that in the absence of any special selective pressure, all plasmids were lost from the culture . The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used . The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5% . In all cases the whole plasmid was lost at a rate higher than 5% . In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such . To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids . The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable . Also in this case, the stability was dependent on the plasmid type and on the growth medium . In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors. J Med Microbiol, 1983 Aug, 16(3), 281 - 93 Effect of serum concentration and metabolic inhibitors on the attachment of Treponema pallidum to rabbit cells; Wong GH et al.; The effect of host-cell metabolism on the attachment of Treponema pallidum to mammalian cells in vitro was studied . The growth of baby rabbit genital organ (BRGO) cells was enhanced by increasing the concentration of serum ("serum shift-up") in the growth medium . Cells starved for 24 h in serum-free medium showed a burst of DNA synthesis when shifted to fresh medium containing 20% serum . In aerobic conditions, they were much more heavily coated with attached T . pallidum than cells shifted to 20% serum after maintenance at serum concentrations of 2.5%, 5% or 10% . This effect was very pronounced during the first few hours of co-incubation . In microaerophilic conditions, the extent of T . pallidum adherence also paralleled the increase in DNA synthesis by BRGO cells . Cycloheximide and methotrexate greatly inhibited DNA and protein synthesis in BRGO cells, but did not affect the motility of T . pallidum . When BRGO cell metabolism was inhibited by these two drugs, attachment of T . pallidum was significantly decreased . These results indicate that T . pallidum attaches best to actively growing BRGO cells in tissue culture . This may explain the apparently preferential parasitism of actively growing tissues by T . pallidum in syphilis in man.