Use of EDTA to prevent spontaneous cell-to-cell transformation provides a more accurate estimation of gene transfer frequencies

Summary Spontaneous cell-to-cell transformation between naturally competent bacteria on selective media resulted in an overestimation of the transferability of genetic information. EDTA effectively prevented transformation on selective media whereas DNaseI did not reliably inhibit cell-to-cell transformation. An improved method to estimate gene transfer frequencies is described.     

The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes

The effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis.     

The localization of tightly bound cardiolipin in cytochrome oxidase

One to two molecules of tightly bound cardiolipin are associated with resolved fractions of cytochrome oxidase containing subunits I to III or I to IV. Large scale isolation of subunits I to IV indicates the presence of approximately 0.5 molecule of cardiolipin per molecule of subunit I. Lipoprotein staining of sodium dodecyl sulfate/urea/acrylamide gels of cytochrome oxidase support the findings that subunit I is a lipoprotein. The resistance of this tightly bound cardiolipin to organic solvent extraction suggests a specific association of some tenacity with the protein.     

Comparison of intestine and bone marrow radiosensitivity of the BALB/c and the C57BL/6 mouse strains and their B6CF1 offspring

The radiosensitivity as measured by LD50/6 or LD50/30 of the F1 hybrid B6CF1 (C57BL/6 X BALB/c) is similar to that of C57BL/6 mice but markedly different from BALB/c. The LD50/6 for BALB/c mice was about 8.8 Gy compared to 16.4 Gy for the B6CF1. The difference in LD50/6 between the parent strains or between BALB/c and the F1 hybrid could not be explained by any differences in crypt cell number, cell cycle time, or transit time. Likewise, the observed differences in the LD50/6 do not appear to result from marked differences in the radiosensitivity of marrow stem cells (CFU-S) since the D0's for the three genotypes of mice were similar. Also, there were no apparent differences in the red blood cell contents of several enzymes associated with antioxidant defenses. The microcolony assay was used to determine the D0 for the crypt clonogenic cells and the D0 values for 60Co gamma rays were about 0.8 Gy for BALB/c mice and 1.4 Gy for B6CF1 mice. However, the D0 values for JANUS fission neutrons were similar; 0.6 Gy for the BALB/c mice and 0.5 for the B6CF1 mice. A comparison of clonogenic cell kinetics, using prolonged colcemid block to distinguish between slowly and rapidly cycling cells suggest that, normally, the stem cells are slowly cycling in both the BALB/c and the B6CF1 hybrid. However, the stem cells of the B6CF1 appear to go into rapid cell cycle more rapidly than those of the BALB/c following irradiation or prolonged colcemid treatment. The more rapid recovery in intestinal epihelial cell production in the B6CF1 hybrid after irradiation may provide an increased mucosal barrier and may, in part, explain the difference in the response to radiation compared to that in the BALB/c.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.     

Developmental Control of Apiogalacturonan Biosynthesis and UDP-Apiose Production in a Duckweed

Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, d-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed d-[³H]glucuronic acid for 30 minutes, the accumulated UDP-[³H]apiose pool accounted for about 27% of the total phosphorylated [³H]pentose derivatives; in turions, the UDP-[³H]apiose pool accounted for only about 4% of the total phosphorylated [³H]pentose derivatives. We conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.     

Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit.

The possibility of the accidental or deliberate release of genetically engineered microorganisms into the environment has accentuated the need to study their survival in, and effect on, natural habitats. In this study, Pseudomonas putida UWC1 harboring a non-self-transmissible plasmid, pD10, encoding the breakdown of 3-chlorobenzoate was shown to survive in a fully functioning laboratory-scale activated-sludge unit (ASU) for more than 8 weeks. The ASU maintained a healthy, diverse protozoal population throughout the experiment, and the introduced strain did not adversely affect the functioning of the unit. Although plasmid pD10 was stably maintained in the host bacterium, the introduced strain did not enhance the degradation of 3-chlorobenzoate in the ASU. When reisolated from the ASU, derivatives of strain UWC1 (pD10) were identified which were able to transfer plasmid pD10 to a recipient strain, P. putida PaW340, indicating the in situ transfer of mobilizing plasmids from the indigenous population to the introduced strain. Results from plate filter matings showed that bacteria present in the activated-sludge population could act as recipients for plasmid pD10 and actively expressed genes carried on the plasmid. Some of these activated-sludge transconjugants gave higher rates of 3-chlorobenzoate breakdown than did strain UWC1(pD10) in batch culture.     

Does follicular dominance occur in ewes?

The process by which a single follicle is selected to ovulate while others regress is unknown in ewes. If the dominant follicle secretes substances that directly inhibit the growth of other follicles, the superovulatory response to the administration of exogenous gonadotrophins may be blunted. Administration of 1250 iu pregnant mares' serum gonadotrophin (PMSG) before or after the emergence of the dominant follicle in the follicular phase, or 1000 iu PMSG in the presence or absence of a large healthy or atretic follicle during the luteal phase did not affect the induced ovulatory response. Comparisons between the ovary with or without the dominant follicle did not reveal any differences in ovulatory response to PMSG. The in-vitro features (i.e. mitotic index, oestradiol and testosterone production) of follicles ipsilateral or contralateral to the dominant follicle during the early and late follicular phases were also similar. If the dominant follicle secretes substances detrimental to the other follicles, this could be mimicked in vitro. Co-culture of small follicles with the largest follicles in a closed system did not reduce their incorporation of 3H thymidine in granulosa cells, compared with small follicles cultured alone. These data suggest that dominance is probably not operative in sheep. The administration of 500 iu of PMSG during the midfollicular phase increased ovulation rate in Merino ewes, indicating that dominance is essentially passive in ewes and can easily be overcome by raising gonadotrophin concentration.