Antigenic analysis of potato virus A particles and coat protein

Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.     

Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.     

Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

SIRT1 is an NAD⁺-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1.     

Biochemical pathways analysis of microarray results: regulation of myogenesis in pigs

Background  

Combining microarray results and biological pathway information will add insight into biological processes. Pathway information is widely available in databases through the internet.     

Effect of the structure of oligonucleotide substrates on kinetic parameters of interaction with BamHI restrictase

Kinetic values of the BamHI endonuclease interaction with synthetic oligonucleotides, containing some defects, have been determined. These defects were: the absence of the one internucleotide phosphate in the GGATCC sequence; substitution of a phosphate linkage by a methylphosphonate one; 5'-protruding end of the double-stranded oligonucleotide substrate. Some modifications resulted in the increase of the initial rates of cleavage due to higher Vmax values for these substrates. Several structural defects in the oligonucleotide substrates have been shown to intensity the formation of productive complexes with the enzyme, which can be explained by the significant role of the polynucleotide chain kinks in the recognition process. Studies on oligonucleotides with different defects made it possible to reveal the phosphate groups essential for the interaction with BamHI endonuclease.     

The structure and antibiotic sensitivity of the causative agents of community-acquired infectious diseases of bacterial origin in children]

Four hundred and forty pediatric patients at the age of 7 days to 15 years with various infections admitted to the Hospital within a month were examined. The biological material was inoculated to blood agar on the first days of the patient admittance to the Hospital and after the growth the organisms were isolated and identified. Antibiotic susceptibility of the isolates was assayed with the disk diffusion method. 479 strains in all were tested. The most frequent cases requiring hospitalization and antibiotic therapy were those of respiratory tract infections (54.09 per cent), urinary tract infections (26.36 per cent), cutaneous and subcutaneous fat diseases, gastrointestinal diseases and others (about 25 per cent of the cases in all). The main pathogens were Streptococcus viridans, S.aureus and S.epidermidis, as well as Enterobacteriaceae (chiefly E.coli) whose frequencies were practically equal (in 25-35 per cent of the cases). The Pneumococcus isolates amounted to 6.3 per cent. Nonfermenting bacteria (Pseudomonas aeruginosa and Acinetobacter) and some representatives of Enterobacteriaceae (Citrobacter, Serratia, Morganella) were isolated from 7 per cent of the patients. The frequency of Klebsiella and Enterobacter was about 11 per cent. The main pathogens were tested for their susceptibility to amoxycillin/clavulanic acid, ampicillin, oxacillin and gentamicin. The least active antibiotic was ampicillin. 88.8 per cent of the E.coli isolates and 100 per cent of the Klebsiella, P.mirabilis, Morganella, Citrobacter, Enterobacter and Serratia isolates were resistant to it. 53.2 per cent of the Streptococcus isolates including 64.5 per cent of the Pneumococcus isolates were as well resistant to ampicillin. 59.5 per cent of the Streptococcus isolates (mainly S.viridans and Enterococcus) was susceptible to oxacillin, 22.2 per cent of them being moderately susceptible. 62.5 per cent of the Pneumococcus isolates and 78.1 per cent of the Staphylococcus isolates were also susceptible to oxacillin. The highest susceptibility of the isolates was that to amoxycillin/clavulanic acid, i.e. 90.1 per cent of the strains, 79.9 per cent of them being highly susceptible. All the isolates of Citrobacter, Serratia and Morganella and some isolates of P.aeruginosa, Acinetobacter, Enterobacter, Klebsiella and E.coli were resistant to amoxycillin/clavulanic acid. As for the latter 5 organisms their susceptibility to amoxycillin/clavulanic acid was comparable with that to gentamicin. The susceptibility of the Streptococcus and Staphylococcus isolates to amoxycillin/clavulanic acid was significantly much higher than that to oxacillin, gentamicin and ampicillin: 93 per cent of the Streptococcus isolates (62.7 per cent of the Pneumococcus isolates) and 90.7 per cent of the Staphylococcus isolates.