Relationship between DNA alkylation and specific-locus mutation induction by N-methyl- and N-ethyl-N-nitrosourea in cultured Chinese hamster ovary cells (CHO/HGPRT system)

Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43--13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per mumol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7--26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per mumol nucleotide per mM. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringently-defined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01--2.0 mM); mean values were 2800 and 840 mutants per 10(6) clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.     

Effects of bile salts on the plasma membranes of isolated rat hepatocytes.

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.     

Genetic study of plasmid-associated high-frequency mutations to streptomycin resistance in Escherichia coli]

Escherichia coli CTR1(RT1)RHfm1) carrying two H-factors and having unusually high frequency of mutation to high level streptomycin resistance is studied. The high frequency of mutation (about 10(-6) to streptomycin resistance is connected with the presence of R factor RHfm1, controlling the resistance to chloramphenicol and low level streptomacin resistance, but not with RT1, controlling the resistance to tetracycline. Spontaneous or ethidium bromide-induced loss of RHfm1 is accompanied by a decrease of the mutation frequency to 10(-9). RHfm1 is efficiently transmissible to other strains at 28 degrees C. The acquisition of RHfm1 by strains of E. coli K-12 ans S. typhimurium LT2 was followed by a 1000--10000-fold increase of the frequejcy of mutation to streptomycin resistance. Some streptomycin resistant mutants were isolated, and chromosome location of the mutations was demonstrated. The streptomycin resistant mutants were unable to transmit high level of resistance to streptomycin with R factor, but only low level one. The loss of RHfm1 by streptomycin resistant mutants was accompanied by the return to the streptomycin sensitivity of the initial R- strans (E. coli K-12 mutants) or by a decrease of the streptomycin resistance to the level, only 2-fold higher than that of R- wild type (E. coli CTR1 mutant). Thus, the mutantions had practically no effect on streptomycin resistance of R- strains, but could lead to high resistance phenotypes in the presence of RHfm1. The mutant loci in all three studied strains were found to be closely linked to the locus "fus" on the genetic map of E. coli.     

Effects of proteinase inhibitors on adenylate cyclase.

The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.     

Allelopathic activity of luteolin 7-O-β-glucuronide isolated from Chrysanthemum morifolium L.

Two flavones, luteolin 7-O-β-glucuronide and diosmetin 7-O-β-glucuronide, were isolated and identified from Chrysanthemum morifolium L. v. Ramat leaves. Identification techniques included HPLC DAD, MS, ¹H and ¹³C NMR spectroscopy. At concentrations of 0.2 and 2.0 mM, luteolin 7-O-β-glucuronide significantly reduced the frond number and chlorophyll content of Lemna gibba plants, but did not significantly affect dry weight. At a concentration of 0.2 mM diosmetin 7-O-β-glucuronide had no significant effect on frond number, dry weight or chlorophyll concentration of L. gibba. These results indicate that an ortho-3′,4′-dihydroxy arrangement of the B-flavonoid ring in the luteolin compound is probably responsible for allelopathic activity.     

Rat and guinea pig pancreatic acini possess both VIP(1) and VIP(2) receptors, which mediate enzyme secretion

Pancreatic acini from most species possess vasoactive intestinal peptide (VIP) receptors. Recently, two subtypes of VIP receptors, VIP(1)-R and VIP(2)-R, were cloned. Which subtype exists on pancreatic acini or mediates secretion is unclear. To address this, we examined pancreatic acini from both rat and guinea pig. VIP(1)-R and VIP(2)-R mRNA were identified in dispersed acini from both species by Northern blot analysis and in rat by Southern blot analysis. With the use of the VIP(2)-R-selective ligand Ro-25-1553 in both species, inhibition of binding of (125)I-labeled VIP to acini showed a biphasic pattern with a high-affinity component (10%) and a second representing 90%. The VIP(1)-R-selective ligand, [Lys(15),Arg(16),Leu(27)]VIP-(1-7)-GRF-(8-27), gave a monophasic pattern. Binding of Ro-25-1553 was better fit by a two-site model. In both rat and guinea pig acini, the dose-response curve of Ro-25-1553 for stimulation of enzyme secretion was biphasic, with a high-affinity component of 10-15% of the maximal secretion and a low-affinity component accounting for 85-90%. At low concentrations (10 nM) of Ro-25-1553 and [Lys(15),Arg(16), Leu(27)]VIP-(1-7)-GRF(8-27), which only occupy VIP receptors, a 4-fold and a 56-fold increase in cAMP occurred, respectively. These results show that both VIP(1)-R and VIP(2)-R subtypes exist on pancreatic acini of rat and guinea pig, their activation stimulates enzyme secretion by a cAMP-mediated mechanism, and the effects of VIP are mediated 90% by activation of VIP(1)-R and 10% by VIP(2)-R. Because VIP has a high affinity for both VIP-R subtypes, its effect on pancreatic acini is mediated by two receptor subtypes, which will need to be considered in future studies of the action of VIP in the pancreas.     

Inhibition of the development of the cellular slime mould Dictyostelium discoideum by omega-aminocarboxylic acids.

Four omega-aminocarboxylic acids - epsilon-aminocaproic acid (EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), p-aminomethylbenzoic acid (PAMBA) and omega-aminocaprylic acid (OACA) -- prevented fruiting body formation of the cellular slime mould Dictyostelium discoideum. At concentrations of 40 mM, 75 mM, 10 mM and 5 mM, respectively, they allowed aggregation but prevented all further development at 24 degrees C. At lower concentrations, EACA allowed fruiting body formation but with a reduced number of spores per fruiting body. Only t-AMCHA had a significant inhibitory effect on the growth of myxamoebae. EACA affected development only if it was present between 8 and 16 h after the cells were deposited on the filters. Its effect was enhanced by high salt concentrations and by higher temperature, and was also dependent on the manner in which the cells were grown. Only strains capable of axenic growth displayed this sensitivity to EACA, although strains carrying only one of the genetic markers for axenic growth (axe A) were partially sensitive.     

Thiol-peptide level and proteomic changes in response to cadmium toxicity in Oryza sativa L. roots

In the present study, rice seedlings were exposed to a range of Cd concentrations (0.1 μM, 1 μM, 10 μM, 100 μM and 1 mM) for 15 days and a combination of different molecular approaches were used to evidence Cd effects and to assess the plants’ ability to counteract metal toxicity. At a macroscopical level, only the highest Cd concentration (1 mM) caused a complete plant growth inhibition, whereas the lowest concentrations seemed to stimulate growth. At genome level, the amplified fragment length polymorphism (AFLP) technique was applied to detect DNA sequence changes in root cells, showing that all the Cd concentrations induced significant DNA polymorphisms in a dose-dependent manner. Data also evidenced the absence of preferential mutation sites.Plant responses were analysed by measuring the levels of gluthatione (GSH) and phytochelatins (PCs), the thiol-peptides involved in heavy metal tolerance mechanisms. Results showed a progressive increase of GSH up to 10 μM of Cd treatment, whereas a significant induction only of PC₃ was detected in roots of plants exposed to 100 μM of Cd. As suggested by the proteome analysis of root tissues, this last concentration strongly induced the expression of regulatory proteins and some metabolic enzymes. Furthermore, the treatment with 10 μM of Cd induced changes in metabolic enzymes, but it mainly activated defence mechanisms by the induction of transporters and proteins involved in the degradation of oxidatively modified proteins.     

R-plasmids of bacteria of the genus Pseudomonas isolated from industrial sewage

A total of 132 Pseudomonas strains isolated from untreated sewage of antibiotic plants were tested. A significant number of the strains were resistant to streptomycin (77 per cent), carbenicillin (75 per cent), kanamycin (37.5 per cent) and tetracycline (23 per cent). Eighteen conjugative and 3 nonconjugative resistance plasmids were detected in 19 strains. The genes determining the resistance to streptomycin, kanamycin and tetracycline were most frequent. The frequency of the plasmid transfer between the strains of Ps. aeruginosa (PAO) varied within 10(-3)--10(-7) per donor cell. Six plasmids belonged to group Inc P-1. Four plasmids belonged to group Inc P-2, 3 plasmids to groups Inc P-3 and Inc P-5 and 1 plasmid to group Inc P-7.     

Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast.     

Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine resistant mutations by phloxine, a mutagenic red dye.

Disodium 9-(3',4',5',6'-tetrachloro-o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 mug/ml. At concentrations of more than 30 mug/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2 h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 mug/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 mug/ml of phloxine increased the frequency to six times that in untreated cultures.     

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.     

Influence of saline irrigation on growth,ion accumulation and partitioning,and leaf gas exchange of carrot (Daucus carota L.)

Like those of many horticultural crop species, the growth and leaf gas exchange responses of carrot (Daucus carota L.) to salinity are poorly understood. In this study ion accumulation in root tissues (periderm, xylem and phloem tissues) and in leaves of different ages was assessed for carrot plants grown in the field with a low level of salinity (5.8 mM Na(+) and 7.5 mM Cl(-)) and in a glasshouse with salinity ranging from 1-80 mM. At low levels of salinity (1-7.5 mM), in both the field and glasshouse, carrot leaves accumulated high concentrations of Cl(-) (140-200 mM); these appear to be the result of a high affinity for Cl(-) uptake and a low retention of Cl(-) in the root system. However, Cl(-) uptake is under tight control, with an 80-fold increase in external salinity resulting in only a 1.5-fold change in the Cl(-) concentration of the shoot and no increase in the Cl(-) concentration of the root xylem tissue. In contrast to Cl(-), shoot Na(+) concentrations were comparatively low (30-40 mM) but increased by seven-fold when salinity was increased by 80-fold. Growth over the 56-d treatment period in the glasshouse was insensitive to salinity less than 20 mM, but at higher concentrations the yield of carrot tap roots declined by 7 % for each 10 mM increase in salinity. At low levels of salinity the accumulation of high concentrations of Cl(-) (150 mM) in carrot laminae did not appear to limit leaf gas exchange. However, photosynthesis and stomatal conductance were reduced by 38 and 53 %, respectively, for plants grown at a salinity of 80 mM compared with those grown at 1 mM. Salinity-induced reductions in both p(i) and carbon isotope discrimination (delta) were small (2.5 Pa and 1.4 per thousand, respectively, at 80 mM) indicating that the reduction in photosynthesis was only marginally influenced by CO(2) supply. At a salinity of 80 mM the photosynthetic capacity was reduced, with a 30 % reduction in the CO(2)-saturated rate of photosynthesis (A(max)) and a 40 % reduction in both the apparent rate of RuBP-carboxylase-limited CO(2) fixation (V(cmax)) and the electron transport rate limiting RuBP regeneration (J(max)). This study has shown that carrot growth and leaf gas exchange are insensitive to the high leaf Cl(-) concentrations that occur at low levels (1-7 mM) of salinity. However, growth is limited at salinity levels above 20 mM and leaf gas exchange is limited at salinity levels above 8 mM.     

Aminoglycoside antibiotics affect hippocampal LTP: a comparative study with the N-type calcium antagonist omega-conotoxin-GVIA.

The in vitro activity of N-type calcium antagonists such as omega-conotoxin-GVIA and the aminoglycoside antibiotics neomycin and streptomycin was studied in rat hippocampal slices. The effects of the drugs were tested on basal CA1 synaptic transmission and on the hippocampal long-term potentiation (LTP) induced by tetanic electrical stimulation and by increasing (4mM) the calcium concentration. Omega-conotoxin-GVIA, neomycin and streptomycin were able to significantly reduce the amplitude of the CA1 population spike at 1 microM, 0.5 mM and 1 mM, respectively. In addition, the drugs affected the induction and maintenance of the CA1 tetanic and calcium-induced LTP at concentrations which did not modify the magnitude of the control CA1 population spike. Omega-conotoxin-GVIA (0.5 microM), neomycin (0.3 mM) and streptomycin (0.7 mM) perfused for 60 min, before inducing LTP, prevented the subsequent increase of the CA1 population spike in all the experiments. The same concentrations of these drugs perfused for 60-min after a previously established LTP significantly reduced the amplitude of the CA1 population spike. The results promote a role for the N-type calcium channels and for the release of neurotransmitters in both the induction and the maintenance of hippocampal LTP.     

Aminoglycoside-Ca++ interactions in skeletal muscle preparations.

Neomycin and streptomycin are equally potent in inhibiting rates of Ca uptake by skeletal muscle fragmented sarcoplasmic reticulum (FSR) and also by heavy FSR isolated between 2000–8000 × G, and affect both membrane preparations to the same extent. In media containing initial free Ca concentrations of 10⁻⁷ and 10⁻⁶M (pCa 7, 6) the inhibition by neomycin of FSR Ca uptake rate is dependent upon the pCa and appears to be competitive, the antibiotic concentrations producing half-maximal effects on Ca uptake being 0.075mM at pCa and 0.53mM at pCa 6. Both MgATPase and CaATPase of FSR are inhibited by neomycin but Ca efflux from FSR partially loaded with Ca oxalate is increased by the antibiotic. Neomycin is 3.2 times as potent as streptomycin in producing 50% neuromuscular blockade in rat phrenic nerve-diaphragm preparations. The ED₅₀ for blockade by neomycin is 0.23mM in medium containing 1.3mM total Ca, but 0.99mM neomycin is required when the medium total Ca is raised to 2.6mM, although the dose-response curves are parallel. Minimal penetration of neomycin into the muscle cell and undetectable effect on the sarcoplasmic reticulum $\text{in}$$\text{situ}$ is indicated by the small decrease in contractility and unchanged relaxation time after exposure to 4.88mM antibiotic, i.e. 20 times the ED₅₀ for neuromuscular blockade.     

Bacillus subtilis mutants dependent on streptomycin

Summary Six streptomycin-dependent mutants of Bacillus subtilis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.     

The mutagenic activity of hydroxyurea in Chlamydomonas reinhardi.

Hydroxyurea, an inhibitor of ribonucleotide reductase, increases the frequency of streptomycin resistant mutants in liquid cultures of Chlamydomonas reinhardi after 45 h incubation. After more prolonged incubation in hydroxyurea medium the frequency of streptomycin resistant mutants declines. This may be due to the slower growth rate of streptomycin resistant mutants compared to wild type cells in hydroxyurea containing medium. Studies on solid medium show that both the rate of forward mutation to streptomycin resistance and reverse mutation to nicotinamide independence are increased several fold by growth on hydroxyurea.     

Acetaldehyde affects mammalian neuromuscular transmission without observable postsynaptic effects.

Acetaldehyde, the first metabolite of ethanol, caused a reversible block of the end-plate potential (EPP) in the rat and mouse phrenic nerve--hemidiaphragm preparation. Decrease and block of the EPP occurred over a bath concentration range from 3 to 25 mM. The phrenic nerve compound action potential was blocked along with the EPP, and this block was not reversed by high bath Ca2+ concentration. The muscle action potential was unaffected even at concentrations up to 50 mM. Over the same concentration range (3--25 mM), miniature end-plate potential (MEPP) frequency sometimes decreased a few minutes after application, and over the ensuing 10--20 min would steadily increase to as much as 11 times the base-line frequency, particularly with higher doses. However, the shape of averaged MEPPs remained unchanged after acetaldehyde application, suggesting that this aldehyde does not have post-synaptic effects.     

Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.     

Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells.

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.