The effects of neuropeptide Y on skeletal muscle contractile properties in streptozotocin diabetic rats

Diabetes induces changes in the structural, biochemical, electrical, and contractile properties of skeletal muscles. Neuropeptide Y (NPY) administered locally can induce angiogenesis in a rat ischemic limb model and restore the contractile function of the ischemic muscle. The effects of NPY on the contractile characteristics of limb skeletal muscles were examined in streptozotocin-induced diabetic rats. Rats were treated with sham pellets (control groups) or NPY-containing pellets (1 mg of NPY/pellet, 14 days releasing time) administered locally to the rat hind limb 2 months after induction of diabetes. Contractile properties and fatigability of the slow-twitch soleus and fast-twitch gastrocnemius medials muscle were compared in control (sham), control NPY, diabetic (sham), and diabetic NPY groups. In order to induce fatigue trains of repetitive tetanic stimulation were used (600 ms/1 s simulation-rest cycle per train, 112 trains at an 85-Hz fusion frequency). Two months of untreated diabetes significantly prolonged soleus contraction and slowed its relaxation, but had minimal effects on soleus tension. NPY ameliorated the diabetic effects on soleus speed-related contractile properties, restoring its contraction and relaxation times. Diabetes significantly reduced gastrocnemius medials tetanic tension, leaving its contractile characteristics mostly unaffected. NPY partially restored gastrocnemius tetanic tension production capacity. Diabetes significantly increased fatigability of both muscles, which was partially restored by NPY, as evidenced by restored endurance of soleus muscle. The results suggest that NPY administered locally tends to normalize muscle performance and improve fatigue resistance of skeletal muscles in streptozotocin diabetes. Further examination is needed to establish the mechanisms of local NPY action on muscle contractile properties in streptozotocin-induced diabetes.     

Isolation and characterization of enteroaggregative,enterotoxigenic, diffusely adherent <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> Worthington from human diarrhoeic faecal samples in Kashmir and Secunderabad,India

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir.     

N-acetyltransferase polymorphism among northern Sudanese

Interindividual and interethnic differences in allele frequencies of N-acetyltransferase (NAT2) single nucleotide polymorphisms (SNPs) are responsible for phenotypic variability of adverse drug reactions and susceptibility to cancer. We genotyped the seven NAT2 common SNPs in 127 randomly selected unrelated northern Sudanese subjects using allele-specific and RFLP polymerase chain reaction (PCR) based methods. Molecular genotyping was enough to designate alleles for 41 individuals unambiguously, whereas 63 individuals' alleles were inferred from haplotypes previously described. In the remaining 23 individuals, however, the phase of the SNPs could not be decided because of multiple SNP heterozygotes. Using computational methods in the HAP and Phase programs, we confirmed the inferred alleles of the 62 individuals and predicted the remaining 23 ambiguous alleles. Twelve NAT2 alleles were identified. Four alleles coded for rapid acetylators (18%), and eight alleles coded for slow acetylators (82%). Two genotypes coded for rapid acetylation (3.9%), 10 for intermediate acetylation (27.6%), and 13 for slow acetylation (68.5%). The G191A African SNP and the G857A predominantly Asian SNP were each detected at a low frequency of 3.1%. The combination of molecular and computational analysis was useful in resolving ambiguous genotypes of NAT2 in multiple SNP heterozygotes. Among the northern Sudanese the SNPs associated with slow acetylation are more prevalent than in Caucasians and Asians. This and other African studies are suggestive of an African origin for NAT2-associated polymorphism.     

Cholinergic anti-inflammatory pathway activity and High Mobility Group Box-1 (HMGB1) serum levels in patients with rheumatoid arthritis

High Mobility Group Box-1 (HMGB1) is a cytokine implicated in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. The cholinergic anti-inflammatory pathway, a vagus nerve-dependent mechanism, inhibits HMGB1 release in experimental disease models. Here, we examine the relationship between vagus nerve activity and HMGB1 in patients with RA. We compared RR interval variability, an index of cardiac vagal modulation, HMGB1 and hsCRP serum levels, and disease activity scores in thirteen RA patients and eleven age- and sex-matched controls. In RA patients, serum levels of HMGB1 and hsCRP were elevated as compared with controls (HMGB1=71 ng/mL [45-99] vs. 18 ng/mL [0-40], P<0.0001; hsCRP=14.5 mg/L [0.7-59] vs. 1 mg/L [0.4-2.9], P<0.001). RR interval variability in RA patients was significantly decreased as compared with controls (HF=38 msec2 [14-80] vs. 288 msec2 [38-364], P<0.0001; rMSSD=20.9+/-9.79 msec, 52.6+/-35.3 msec, P<0.01). HMGB1 levels and RR interval variability were significantly related (rho=-0.49, P<0.01). HMGB1 serum levels significantly correlated with disease activity scores (DAS-28) in patients with RA (P=0.004). The study design does not enable a determination of causality, but the results are consistent with the hypothesis that decreased cholinergic anti-inflammatory pathway activity is associated with increased HMGB1 levels in patients with RA.     

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS

Introduction – Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. Objective – This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. Methodology – The specific focus of the study is on thin‐layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf‐MELDI‐MS). Samples employed in the study were standards and microwave‐assisted water extracts from Quercus. Results – TLC analysis proved the presence of mono‐, di‐ and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC‐MS confirmed data obtained via TLC for mono‐ to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf‐MELDI‐MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf‐MELDI‐MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Conclusion – Evaluation of all three techniques employed, clearly proved the heightened performance of mf‐MELDI‐MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC‐MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin-proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators.     

Occult HCV or delayed viral clearance from lymphocytes of Chronic HCV genotype 3 patients after interferon therapy

Background

A recently discovered occult HCV entity reported by various investigators seems to be highly controversial. Especially, the clinical significance of these findings remains uncertain. For optimal outcome of antiviral therapy, investigation of occult HCV needs a broad-based probe in order to investigate the results of viral therapy and its host/viral interaction. The current study was aimed at determining the prevalence of occult HCV in peripheral blood lymphocytes of predominantly genotype 3 HCV-infected patients after completion of antiviral therapy and to investigate long term outcomes in the presence or absence of PBMC positivity.

Method

A total of 151 chronic, antiHCV and serum RNA-positive patients were enrolled in the study. Patients with a complete virological response at the end of treatment were screened for the presence of viral RNA in their PBMCs and were followed for up to one year for the presence of serum and PBMC viral genomic RNA.

Results

Out of 151 patients, 104 (70%) responded to the prescribed interferon treatment and showed viral-clearance from serum. These were screened for the presence of genomic RNA in their PBMCs. Sixteen samples were PBMC-positive for viral RNA at the end of treatment (EOT). All these patients had also cleared the virus from peripheral blood cells after the 6-12 month follow-up study.

Conclusion

True occult hepatitis C virus does not exist in our cohort. Residual viremia at the EOT stage merely reflects a difference in viral kinetics in various compartments that remains a target of immune response even after the end of antiviral therapy and is eventually cleared out at the sustained viral response (SVR).     

Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101

Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 gL(-1) starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h(-1) and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0 gL(-1) total ABE. Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2 gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2 gL(-1) ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.