Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes.     

Peptidomic analysis of HEK293T cells: effect of the proteasome inhibitor epoxomicin on intracellular peptides

Peptides derived from cytosolic, mitochondrial, and nuclear proteins have been detected in extracts of animal tissues and cell lines. To test whether the proteasome is involved in their formation, HEK293T cells were treated with epoxomicin (0.2 or 2 μM) for 1 h and quantitative peptidomics analysis was performed. Altogether, 147 unique peptides were identified by mass spectrometry sequence analysis. Epoxomicin treatment decreased the levels of the majority of intracellular peptides, consistent with inhibition of the proteasome beta-2 and beta-5 subunits. Treatment with the higher concentration of epoxomicin elevated the levels of some peptides. Most of the elevated peptides resulted from cleavages at acidic residues, suggesting that epoxomicin increased the processing of proteins through the beta-1 subunit. Interestingly, some of the peptides that were elevated by the epoxomicin treatment had hydrophobic residues in P1 cleavage sites. Taken together, these findings suggest that, while the proteasome is the major source of intracellular peptides, other peptide-generating mechanisms exist. Because intracellular peptides are likely to perform intracellular functions, studies using proteasome inhibitors need to be interpreted with caution, as it is possible that the effects of these inhibitors are due to a change in the peptide levels rather than inhibition of protein degradation.     

Low efficacy of clarithromycin including sequential regimens for Helicobacter pylori infection

Background: Sequential treatment for Helicobacter pylori (H. pylori) appears to achieve a better eradication rate than triple therapy. However, most of the data have been reported from the Italy, and studies from different population are needed before it is recommended in clinical practice. The present study aimed to assess and compare the efficacy of two separate clarithromycin including sequential regimens in Turkey which is well known with high clarithromycin and metronidazole resistance to H. pylori. Methods: Consecutive H. pylori ‐positive patients with non‐ulcer dyspepsia were randomly allocated to one of the two sequential regimens; the first group was given lansoprazole 30 mg b.i.d. plus amoxicillin 1 g b.i.d. for the first week, followed by lansoprazole 30 mg b.i.d., clarithromycin 500 mg b.i.d., and metronidazole 500 mg t.i.d. for the second week (LA‐CM). The second arm was given the same regimen but tetracycline500 g q.i.d. instead of metronidazole (LA‐CT). H. pylori was detected with urea breath test (UBT) and histology before enrollment. UBT was repeated at 6th weeks after treatment. Results: A total of 200 patients were enrolled in groups and 179 of them completed their protocols. The cumulative per protocol (“PP”) and intention‐to‐treat (“ITT”) eradication rates were 74.3% and 66.5% in all patients, respectively. Both “PP” (78.2% vs 70.1%) and “ITT” (72% vs 61%) eradication rates were better in LA‐CT group than LA‐CM group, but the differences were not statistically significant (p > .05). Both regimens were well tolerated, and the incidence of adverse effects was comparable. Conclusion: Two weeks clarithromycin including sequential regimens with metronidazole or tetracycline were not achieved acceptable eradication rates in Turkey.     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 μg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.     

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.     

Arsenical resistance in the IncHI2 plasmids

The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100mM) and sodium arsenite (up to 10mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis.     

Seed selection for grassland restoration: competitive effect of a dominant grass is mediated by seed source and nutrient availability

Cultivars are selected for advantageous traits, which may enable them to have a greater competitive effect (CE), particularly under high nutrient conditions. Sites with high nutrient availability may favor the development of dominant grasses rather than subordinate species, especially in nutrient‐limited systems, such as calcareous grasslands. The aim of this experiment was to determine whether seed source influences the CE of a dominant grass on a subordinate species, and whether this relationship is mediated by nutrient availability. A greenhouse experiment with three nutrient levels was established; Dianthus carthusianorum was chosen as the subordinate “phytometer” to detect variation in the CE of different Festuca rubra seed sources. The grass species was sourced from 13 cultivar and 12 commercially propagated, but not selected, wild sources. When CE was calculated from biomass, propagated wild seed sources of F. rubra had a greater CE on the subordinate species than cultivars for medium and high nutrient levels. Based on phytometer height, propagated wild seed sources of F. rubra had a greater CE under all three nutrient levels. Our findings do not support the general notion that cultivars are more competitive than wild genotypes. Thus, the cultivar F. rubra may facilitate the establishment of other species during grassland restoration, particularly under elevated nutrient conditions.