Location and interactions of phospholipid and cholesterol in human low density lipoprotein from 31P nuclear magnetic resonance.

The major phospholipids, phosphatidylcholine and spingomyelin, of low density lipoprotein (LDL) are accessible to small amounts of Pr3+, suggesting that the head groups of all mobile phospholipids are on the surface of the particle in contact with the aqueous medium. The major source of the nuclear Overhauser effect enhancement of 31P resonances is the N-methyl proton of the choline moiety, indicating close N-methyl phosphate group interactions, probably similar to those found previously in phospholipid vesicles. This behavior of the phospholipid head groups in LDL is similar to that in small vesicles without cholesterol, suggesting that in LDL most of the cholesterol is not associated with mobile, surface phospholipids. In contrast to LDL, where the presence of a large protein immobilizes some phospholipid head groups, immobilization does not occur in high density lipoprotein, consistent with occurrence of smaller peptides in the latter.     

Antibiotic resistance and pathogenicity factors in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from mastitic Sahiwal cattle

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. In the present study, the distribution of mastitic MRSA and antibiotic resistance was studied in 107 strains of S. aureus isolated from milk samples from 195 infected udders. The characterizations pathogenic factors (adhesin and toxin genes) and antibiotic susceptibility of isolates were carried out using gene amplification and disc diffusion assays, respectively. A high prevalence of MRSA was observed in the tested isolates (13.1%). The isolates were also highly resistant to antibiotics, i.e. 36.4% were resistant to streptomycin, 33.6% to oxytetracycline, 29.9% to gentamicin and 26.2% each to chloramphenicol, pristinomycin and ciprofloxacin. A significant variation in the expression of pathogenic factors (Ig, coa and clf) was observed in these isolates. The overall distribution of adhesin genes ebp, fib, bbp, fnbB, cap5, cap8, map and cna in the isolates was found to be 69.1, 67.2, 6.5, 20.5, 60.7, 26.1, 81.3 and 8.4%, respectively. The presence of fib, fnbB, bbp and map genes was considerably greater in MRSA than in methicillin-susceptible S. aureus (MSSA) isolates. The proportions of toxin genes, namely, hlb, seb, sec, sed, seg and sei, in the isolates were found to be 94.3, 0.9, 8.4, 0.9, 10.2 and 49.5%, respectively. The proportions of agr genes I, II, III and IV were found to be 39.2, 27.1, 21.5 and 12.1%, respectively. A few isolates showed similar antibiotic-resistance patterns, which could be due to identical strains or the dissemination of the same strains among animals. These findings can be utilized in mastitis treatment programmes and antimicrobials strategies in organized herds.     

SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation

SV40 large T antigen has been reported to be modified with several different sugars including N-acetylglucosamine, galactose, and mannose. In this report we have reexamined the glycosylation of T antigen and found that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein. Surprisingly, even though [3H]galactose could be metabolically incorporated into the protein, analysis showed that all of the radioactivity in T antigen had been converted to other species. The N-acetylglucosamine was demonstrated to be linked to the protein in the form of O-linked N- acetylglucosamine, the best characterized form of nuclear and cytoplasmic glycosylation in mammalian systems. We have localized the major site of glycosylation to the amino terminal portion of the molecule. Analysis of mutated T antigen where serines 111/112 were substituted with alanine suggest that these residues constitute a glycosylation site on the protein. These two serines fall within a typical O-linked N-acetylglucosamine glycosylation site (PSS) and are also known to be phosphorylated. Thus, it is likely that competition between phosphorylation and glycosylation occurs at this site.      

Docking studies on novel analogues of 8 methoxy fluoroquinolones against GyrA mutants of Mycobacterium tuberculosis

Background

Modeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues.

Results

Using sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rβ and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus.

Conclusions

We succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.