G2 effects of DNA-repair inhibitors on chromatid-type aberrations in root-tip cells treated with maleic hydrazide and mitomycin C

In recent years the existence of a DNA-repair process in G2 has been proposed to explain the potentiating effects of DNA-repair inhibitors given in G2 on chromatid aberrations (CA) induced by S-dependent as well as S-independent DNA-damaging agents. In the present report, root-tip cells of Allium cepa were exposed to maleic hydrazide (MH) or mitomycin C (MMC) and post-treated in G2 with caffeine (Caff) and various inhibitors of DNA synthesis. No enhancement of chromosome damage was observed when Caff was present in G2, but hydroxyurea (HU) or 5-fluorodeoxyuridine (FdUrd) potentiated the frequencies of CA. A slight additional increase of CA frequencies was observed following treatment with Ara C and excess thymidine in G2. When MH-damaged cells were pulse-treated with Caff earlier during recovery, the yield of CA was enhanced. The earlier Caff was present following MH treatment, the stronger was the potentiation.     

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Summary Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.Abbreviations Tc tetracycline - Sp spectinomycin - Rif rifampicin - Km kanamycin     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Comparative effects of tumour necrosis factor-alpha (cachectin), interleukin-1-beta and tumour growth on amino acid metabolism in the rat in vivo. Absorption and tissue uptake of alpha-amino[1-14C]isobutyrate.

Incubation of a membrane preparation enriched in Photosystem Two (PSII) at alkaline pH inhibited the water-splitting reactions in two distinct steps. Up to pH 8.5 the inhibition was reversible, whereas at higher alkalinities it was irreversible. It was shown that the reversible phase correlated with loss and rebinding of the 23 kDa extrinsic polypeptide. However, after mild alkaline treatments a partial recovery was possible without the binding of the 23 kDa polypeptide when the assay was at the optimal pH of 6.5 and in a medium containing excess Cl-. The irreversible phase was found to be closely linked with the removal of the 33 kDa extrinsic protein of PSII. Treatments with pH values above 8.5 not only caused the 33 kDa protein to be displaced from the PSII-enriched membranes, but also resulted in an irreversible modification of the binding sites such that the extrinsic 33 kDa protein could not reassociate with PSII when the pH was lowered to 6.5. The results obtained with these more extreme alkaline pH treatments support the notion that the 23 kDa protein cannot bind to PSII unless the 33 kDa protein is already bound. The differential effect of pH on the removal of the 23 kDa and 33 kDa proteins contrasted with the data of Kuwabara & Murata [(1983) Plant Cell Physiol. 24, 741-747], but this discrepancy was accounted for by the use of glycerol in the incubation media.     

Effect of prenatal exposure to alcohol on membrane-bound enzymes during astrocyte development in vivo and in primary culture

In the present work we have analyzed the effect of prenatal ethanol exposure on the activity of several glial marker and functional enzymes during the development of astrocytes isolated from rat brain as well as in primary culture. The activity of marker enzymes glutamine synthetase and butylcholinesterase showed no differences between isolated astrocytes from 15 and 70 day old control rats. However, the activity of the membrane-bound enzymes (Na+K)ATPase and 5'-nucleotidase was higher in astrocytes from 70 day old control rats than in those from 15 day old animals. Although the pattern found in astrocytes from alcohol-exposed rats was similar to that of controls, the levels of activity of the enzymes were lower in alcoholic than in control animals. When control astrocytes in primary culture were used, the activity of (Na+K)ATPase and 5'-nucleotidase increased throughout the entire culture period. In contrast, the maximal activity of glutamine synthetase was found at 7 days of culture. Ethanol also induced a decrease in the activity of all enzymes, which was more evident at the end of the culture period. These results indicate that the activity of the enzyme markers analyzed increased mainly during the first weeks of life and remained constant after this period. By contrast, the membrane-bound enzymes studied showed a progressive increase with age. In conclusion, since these astrocyte enzymes are important in the regulation of several neuronal functions through the control of the composition of extracellular fluid, the effect of ethanol on their activities could explain some of the neuronal alterations reported in children and animals exposed to ethanol during development.     

Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Identification of the spI products of Balbiani ring genes in Chironomus thummi

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.Abbreviations BR Balbiani ring - spI family of M_r=10⁶ secretory polypeptides     

Performance of nymphal and adult Nezara viridula on selected alternate host plants

Laboratory studies with the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae), feeding on seeds or fruits of some alternate host plants, demonstrated differences in nymphal survival and development time, and body weight at adult emergence. Lowest mortalities (25%) were observed for nymphs on fruits of dakota mustard [Brassica kaber (DC.)] and siberian motherwort (Leonurus sibiricus L.). On fruits of castor bean (Ricinus communis L.) nymph mortality was ca. 60%, and on mature seeds of L. sibiricus ca. 75%. Total development time from second instar to adult ranged from ca. 26 days on B. kaber to ca. 42 days on R. communis. Fresh body weight at adult emergence was greatest for nymphs on L. sibiricus and lowest on R. communis. Adult survival after 30 days and mean longevity was greater on L. sibiricus than on the remaining foods, being drastically reduced on R. communis. In general, reproductive performance on all foods was similar, except R. communis, on which no female reproduced. Female weight gain of N. viridula was generally greater on L. sibiricus On R. communis, adults lost weight during their first week and showed the lowest weight gain at the end of day 29. Field observations coupled with these laboratory studies suggest that in the Warta area (northern Paraná state, Brazil, 23° 11 S), L. sibiricus and B. kaber are important alternate food plants in the seasonal phenology of N. viridula. R. communis, however, is of poor nutritional quality, serving primarily as a refuge.

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Résumé L'alimentation au laboratoire de N. viridula avec les graines et les fruits de diverses plantes a entraîné différentes mortalités larvaires, différentes vitesses de développement et des différences dans les poids des adultes à l'émergence. Les mortalités les plus faibles (25%) on été observées avec les fruits de Brassica kaber DC. et de Leonurus sibiricus L.Avec les fruits de Ricinus communis L., la mortalité larvaire était de 60%; sur graines mûres de L. sibiricus, elle était de 75%. La durée du développement du second stade à l'adulte a varié de 26 jours sur B. kaber à 42 jours sur R. communis. Le poids frais des adultes à l'émergence était le plus élevé avec L. sibiricus et le plus faible avec de R. communis. La longévité des adultes au-delà de 30 jours était la plus élevée avec L. sibiricus, et était fortement réduite avec R. communis. Dans l'ensemble, la reproduction était de même importance avec les différents aliments, à l'exception de R. communis avec lequel les femelles ne se reproduisaient pas. Le gain de poids des femelles était généralement supérieur avec L. sibiricus; avec R. communis les adultes ont perdu du poids pendant la première semaine et ont présenté le plus faible gain à la fin du 29ème jour.Des observations dans la nature, associées à ces expériences de laboratoire, laissent supposer que dans la zone de Warta (au Nord de l'état de Paraná, 23° 11 S, au Bresil), L. sibiricus et B. kaber sont des plantes hôtes alternatives importantes dans la phénologie saisonnière de N. viridula. Cependant, R. communis, de faible valeur alimentaire, sert avant tout de refuge.