Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases.

Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one. Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes. At the other central positions, uracil was eliminated with different initial velocities. Single stranded phi X 174 DNA fragments were used to study the influence of the sequence. Cytosine bases were deaminated to give uracil by bisulfite treatment. It was shown that the initial excision velocity of two vicinal uracil residues was decreased. The same observation was made for two uracils separated by one base. A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.     

Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.     

A metaproteomic approach gives functional insights into anaerobic digestion

Aims: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. Methods and Results: The microbial communities from an anaerobic industrial‐like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum‐like (76%) micro‐organisms. Three hundred and eighty‐eight reproducible protein spots were obtained on 2‐D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO₂ and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro‐organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.     

“Mixed Inhibitors” of HIV-Reverse Transcriptase: Synthesis and Antiviral Activity

Abstract

Some “AZT-HEPT” and “ddC-HEPT” conjugates were designed, synthesized and evaluated for their anti-HIV activity.     

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.     

Synthesis, antimicrobial activity and conformational analysis of novel substituted pyridines: BF(3)-promoted reaction of hydrazine with 2-alkoxy pyridines

Some new 2-alkoxy-3-cyano-4,6-diarylpyridines 3,4 were synthesized by condensation of different alpha,beta-unsaturated ketones 1 with malononitrile 2, followed by cyclization in sodium alkoxide/alcohol system. Lewis acid-catalyzed reaction of 4 with hydrazine afforded the corresponding 1H-pyrazolo[3,4-b]pyridines 5. The potency of the results as antimicrobial agents has been evaluated. The structure of the newly prepared compounds was assessed by microanalysis, IR and NMR spectra. Molecular mechanics (MM2) and semiemperical (AM1) molecular orbital calculations have been performed for the most biologically active compounds 5b and c to get insight into their molecular structures and to learn more about their stable molecular conformations.     

A new specific ageusia: some humans cannot taste L-glutamate

A new specific ageusia was found in human subjects for monosodium L-glutamate (MSG). Four tests were successively applied to discriminate non-tasters and hypotasters from tasters. (i) NaCl and MSG thresholds, and (ii) suprathreshold sensitivity were evaluated using the up-and-down procedure. Only 73% of 109 subjects common to both tests demonstrated a sensitivity for MSG significantly higher than their sensitivity to NaCl, and hence a specific sensitivity to L-glutamate. The remaining 27% who showed no significant difference in sensitivity to MSG and NaCl solutions were considered as putative hypotasters. (iii) Perception profiles (time-intensity) for MSG and NaCl were tested in 58 subjects and appeared significantly different in 47 tasters (81%). This technique helped in identifying among putative hypotasters of tests 1 and 2 a few tasters who perceived equal intensity for isoconcentration of NaCl and MSG but who could discriminate isomolar solutions on other cues. Thus, 19% of subjects, for whom no significant differences were found between MSG and NaCl time-intensity profiles, remained in the hypotaster group. (iv) A discrimination task including 24 triangular presentations per subject of NaCl and MSG 29 mM applied to the eight most severe hypotasters showed that two subjects at least (two of 58; 3.5%) could not discriminate between both stimuli. Moreover, these subjects probably perceived identical sensations for MSG and NaCl solutions. The six other hypotasters (10.3%) could discriminate both stimuli at the limit of significance. None of these eight subjects were able to identify the typical umami taste in 29 mM MSG.     

Corrigendum to "The sow endometrium at different stages of the oestrus cycle: studies on morphological changes and infiltration by cells of the immune system." [Anim. Reprod. Sci. 65 (2001) 95-114

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4+/-0.7 (mean+/-S.D.) were used. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy (LM). The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation om the infiltration and distrobution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells, and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle.     

Carbohydrate cycling in micro-organisms: what can (13)C-NMR tell us?

The extension of (13)C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as 'reversibility', 'metabolic' and 'substrate' cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the (13)C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.