A sensitive immunotransfer method for the detection of oligodendroglial plasma membrane antigens

Efficient transfer of a wide molecular weight range of plasma membrane proteins from polyacrylamide gels to nitrocellulose paper has been achieved. Using published immunodetection procedures (sensitivity: 1 ng protein), two major antigens of molecular weights of 71,000 and 82,000 have been identified in oligodendroglial plasma membranes. This immunotransfer method will be extremely useful for the detection of low levels of antigen in complex membrane protein mixtures.     

Netropsin stimulates the formation of an extracellular proteinase and suppresses protein turnover in sporulating Bacillus megaterium

Abstract Netropsin stimulated the rate of synthesis of an extracellular metalloproteinase in Bacillus megaterium incubated in a sporulation medium. The antibiotic delayed but did not suppress the decrease in the ability to synthesize the proteinase occurring at later sporulation stages. Netropsin also stimulated the synthesis of the proteinase when added to a growing culture; it inhibited the increase of protein turnover which was switched on between the 2nd and 3rd hour in the sporulating population. No refractile spores were developed during 6 h at 35°C in the antibiotic-treated culture. In the control 60% of sporulating cells were observed under similar conditions.     

A single fixation protocol for proteome-wide immunofluorescence localization studies

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.     

Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

ABSTRACT: BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.     

Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin

The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using Dictyostelium discoideum as a model for studying drug resistance showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme that degrades S-1-P, had increased resistance to cisplatin, whereas mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum--sgkA and sgkB--that are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, whereas mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and the SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase-null mutants was reversed by the addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N,N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum-based drug carboplatin but not with doxorubicin, 5-fluorouracil, and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants and should be useful in designing therapeutic schemes involving more than one drug. This study identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum-based drugs.     

Effect of the preweaning nutritional state on the cardiac protein profile and functional performance of the rat heart

The aim of the study was to find out whether the changes in nutritional status induced by different litter size during early postnatal development can influence quantitative and qualitative protein remodeling and contractile performance of the myocardium. Male Wistar rats born at the same day were pooled together at 2 days postbirth and assigned by random selection to dams in groups of 4, 8 or 16 rats/litter. The animals were investigated at the age of 4 and 16 weeks. The results revealed that the early postnatal nutritional modification altered weight parameters: whereas lower heart weight persisted in slow-growing rats until 16 weeks, higher body weight of fast-growing rats returned to the control level at the age of 16 weeks. Altered nutritional status influenced also protein remodeling of the myocardium: the concentration of all noncollagenous proteins (fractions of metabolic and contractile proteins) significantly increased in slow-growing rats, on the other hand, the concentration of collagenous proteins (pepsin-soluble and -insoluble fractions) was higher in fast-growing animals. The changes were, however, only transitional: three months after the end of the weaning period most protein changes returned to the control level. However, higher concentration of total blood lipids and triglycerides in fast-growing rats persisted until adulthood. Nutritional changes had, however, only minor effect on ventricular performance. No differences among groups were observed in basal values of the left ventricular pressure, while the maximum pressure attained after an acute ventricular loading and the contractile reserve were significantly decreased in slowgrowing 4 week old rats. The functional consequence of altered nutritional status during weaning was only transitional, in agreement with the transient character of most structural and biochemical markers of myocardial remodeling.     

The Relation of Hepcidin to Iron Disorders,Inflammation and Hemoglobin in Chronic Kidney Disease

The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by ⁵¹Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m² (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.     

Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds

An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.     

Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows

Cloning technologies, including embryo splitting and nuclear transfer, were introduced into dairy cattle breeding in the early 1980s. With the recent worldwide attention on the cloning of sheep ("Dolly") and cows ("Gene"), the potential food safety concerns for food products derived from cloned animals needs to be addressed. There has been no study of the composition of milk produced by cloned cows. In this preliminary study, we evaluated the composition of milk from 15 lactating non-embryonic cell cloned cows and six non-cloned lactating cows over a single season. The cloned cows came from five unique genetic lines and three distinct breeds. Milk samples were analyzed for total solids, fat, fatty acid profile, lactose, protein and compared to non-cloned and literature values. Gross chemical composition of milk from cloned cows was similar to that of the non-cloned cows and literature values. Our results lead us to conclude that there are no obvious differences in milk composition produced from cloned cows compared to non-cloned cows.