Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

Effect of p-chlorophenylalanine on the reproduction of the rat (author's transl)]

The effect of p-chlorophenylalanie (p-CPA) --300 mg/kg-- on reproduction has been studied in the female rat. Groups of animals were injected with a dose of 300 mg/kg of p-CPA 48 hours before proper copulation conditions at different moments along the ovarian cycle. Presence of spermatozoa in the vaginal frotis was negative in treated rats; in the control groups however, positivity was found in variable proportions according to the phase of the ovarian cycle: 30 ad 90% in diestrus and proestrus respectively. Treated animals showed continuous diestrus phases and diffuse luteinitation of the ovary. The results may indicate that a decrease of cerebral 5-HT, caused by p-CPA, lessens the reproductive behaviour of the female rat through mechanisms depending probably on the liberation of gonadrotrophins.     

Improved localization of thiamine pyrophosphatase activity in mounted cryostat sections using gel fixation and gel incubation media

Summary The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb²⁺; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.     

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.