Strategies for the highly efficient synthesis of erythropoietin N‐glycopeptide hydrazides

A convergent synthesis for erythropoietin (EPO) 1‐28 N‐glycopeptide hydrazides was developed. In this approach, EPO 1‐28 peptides were synthesized on the solid phase and converted to C‐terminal hydrazides after cleavage from the resin. After selective deprotection of the Asp24 side chain, the desired glycosylamine was coupled by pseudoproline‐assisted Lansbury aspartylation. Although the initial yields of the EPO 1‐28 glycopeptides were satisfactory, they could be markedly improved by increasing the purity of the peptide using a reversed‐phase high‐performance liquid chromatography (RP‐HPLC) purification of the protected peptide.     

Unexpected role for granzyme K in CD56bright NK cell-mediated immunoregulation of multiple sclerosis

Functional NK cell deficiencies are associated with autoimmune diseases, including multiple sclerosis. NK cells can promote or inhibit adaptive immunity via either cytokine production or cytotoxicity toward immature dendritic cells and activated T cells. In humans, this immunoregulatory role resides in the CD56(bright) NK cell subset, which is selectively expanded by daclizumab, a CD25-blocking Ab that suppresses multiple sclerosis-associated inflammation. The objective of this study was to investigate the molecular mechanisms underlying the cytotoxicity of NK cells toward activated T cells. We demonstrated that NK cells induce caspase-independent apoptosis that requires NK cell degranulation and causes mitochondrial dysfunction in activated T cells. Although both granzyme A and granzyme K (GrK) can mediate this form of apoptosis, quantitatively we observed preferential transfer of GrK to target cells. Consequently, gene silencing of GrK in the NK-92 cell line, which retains functional characteristics of CD56(bright) NK cells, profoundly inhibited the ability of NK-92 cells to kill activated syngeneic T cells. Finally, we demonstrated that daclizumab treatment significantly enhanced this newly defined mechanism of cytotoxicity by CD56(bright) NK cells. Our study describes the important physiological role that GrK plays in immunoregulation of adaptive immunity in humans and indicates that therapeutic exploitation of this pathway is beneficial in controlling autoimmunity.     

Factorial analysis of the influence of dissolution medium on drug release from carrageenan-diltiazem complexes

This research studied the influence of buffer composition, pH, and ionic strength on the release of diltiazem hydrochloride from a complex of the drug with lambda carrageenan. Two viscosity grades of carrageenan were also compared. A factorial analysis was used to evaluate the influence of individual variables and their interactions. Both the complex solubility, measured as the drug concentration in equilibrium with the solid complex, and the drug release rate from constant surface area were considered. The increase of ionic strength significantly increased complex solubility in all the buffer systems. A significant effect of polymer grade on complex solubility was evidenced only in phosphate buffer with a pH of 6.8, indicating lower solubility of the complex when higher polymer molecular weight was involved. In most cases, drug release rate decreased when high polymer grade was involved in the complex. Ionic strength did not always have a significant effect on drug release rate and was quantitatively less important than for solubility lonic strength especially affected the drug release profiles. At higher ionic strength drug release was no longer constant, but decreased with time, probably because of lower polymer solubility.     

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

Background

Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings.

Methods

Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO_2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NO_x ^-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting.

Results

High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NO_x ^- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels.

Conclusion

The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.     

Marine Bacterioplankton Diversity and Community Composition in an Antarctic Coastal Environment

The bacterial community inhabiting the water column at Terra Nova Bay (Ross Sea, Antarctica) was examined by the fluorescent in situ hybridization (FISH) technique and the genotypic and phenotypic characterization of 606 bacterial isolates. Overall, the FISH analysis revealed a bacterioplankton composition that was typical of Antarctic marine environments with the Cytophaga/Flavobacter (CF) group of Bacteroidetes that was equally dominant with the Actinobacteria and Gammaproteobacteria. As sampling was performed during the decay of sea-ice, it is plausible to assume the origin of Bacteroidetes from the sea-ice compartment where they probably thrive in high concentration of DOM which is efficiently remineralized to inorganic nutrients. This finding was supported by the isolation of Gelidibacter, Polaribacter, and Psychroflexus members (generally well represented in Antarctic sea-ice) which showed the ability to hydrolyze macromolecules, probably through the production of extracellular enzymes. A consistently pronounced abundance of the Gammaproteobacteria (67.8%) was also detected within the cultivable fraction. Altogether, the genera Psychromonas and Pseudoalteromonas accounted for 65.4% of total isolates and were ubiquitous, thus suggesting that they may play a key role within the analyzed bacterioplankton community. In particular, Pseudoalteromonas isolates possessed nitrate reductase and were able to hydrolyze substrates for protease, esterase, and β-galactosidase, thus indicating their involvement in the carbon and nitrogen cycling. Finally, the obtained results highlight the ability of the Actinobacteria to survive and proliferate in the Terra Nova Bay seawater as they generally showed a wide range of salt tolerance and appeared to be particularly competitive with strictly marine bacteria by better utilizing supplied carbon sources.     

LOW FREQUENCY OF MUTATIONS WITH STRONGLY DELETERIOUS BUT NONLETHAL FITNESS EFFECTS

Most experimentally detectable effects of mutations in cellular organisms are either lethal or mildly deleterious. A possible explanation for the paucity of strongly detrimental but nonlethal mutations is that the processes constituting cellular metabolism are either essential or largely redundant. Alternatively, the partition between lethal and inconspicuous mutations exists within important biological processes. To test this, we measured maximum growth rates of yeast strains each carrying the deletion of a single gene in one of 38 protein complexes. We also used relevant data from previous high-throughput phenotypic studies of the yeast gene-deletion collection. The complexes typified well-defined sets of genes engaged in a common process. Within virtually all essential complexes there were two clear modes of phenotypic effects, that is the cessation of growth or slowdown of growth by a few percent. This uniformity is striking given that complexes differ extensively in function, size, and proportion of essential proteins. The pattern of bimodality is observed both under optimal and suboptimal environmental conditions. The generic paucity of strong effects and abundance of small ones relates to the feasibility of analyses of quantitative traits and epidemiological surveys, irrespective of the particular element of metabolism under study.     

Lysosomal thiol reductase negatively regulates autophagy by altering glutathione synthesis and oxidation

Redox regulation is critical for a number of cellular functions and has been implicated in the etiology and progression of several diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. It has been shown that, in the absence of gamma-interferon inducible lysosomal thiol reductase (GILT), cells are under increased oxidative stress with higher superoxide levels and decreased stability, expression, and function of mitochondrial manganese superoxide dismutase (SOD2). Here, we further elucidate the role of GILT in the homeostatic regulation of oxidative stress. We show that GILT-deficient fibroblasts exhibit reduced glutathione levels, shift in GSSG/GSH ratio toward the oxidized form, and accumulate dysfunctional mitochondria. Redox-sensitive pathways involving Erk1/2 activation and nuclear high mobility group box 1 (HMGB1) protein cytosolic translocation are both activated and associated with increased autophagy in GILT−/− fibroblasts. We hypothesize that these events are responsible for degrading the damaged mitochondria and mitochondrial SOD2 in the absence of GILT. This is the first time to our knowledge that a lysosomal enzyme has been implicated in global effects within the cell.     

The non-competitive AMPA receptor antagonist (GYKI 52466) blocks quisqualate-induced acetylcholine release from the rat hippocampus and striatum: an in vivo microdialysis study

The effects of the non-N-methyl-D-aspartate (NMDA) agonist quisqualate (QUIS) and selective AMPA/kainate receptor antagonist 1-(aminophenyl)-methyl-7, 8-methyilendioxy-5H-2,3-benzodiazepine (GYKI 52466) on the release of acetylcholine (ACh) from the hippocampus and striatum of freely moving rats were studied by transversal microdialysis. Acetylcholine level in the dialisate was measured by the high performance liquid chromatography (HPLC) method with an electrochemical detector. The QUIS (100 microM) perfused through the striatum induced an increase of extracellular ACh level (250%) which lasted for over 1h and gradually returned to basal values. Local perfusion of GYKI 52466 (10-100 microM) to the striatum did not change the basal release of ACh. GYKI 52466 (10 microM) administered together with QUIS (100 microM) in he striatum antagonized the stimulant effect of QUIS on the ACh release.Local administration of the QUIS (100 microM) through the microdialysis fiber implanted in the hippocampus, caused a long lasting increase of extracellular hippocampal ACh level (360%) which was reversed when the drug was withdrawn from the perfusion solution. The stimulant effect of QUIS was antagonized by concomitant perfusion of GYKI (10 microM). No effect was seen on the basal ACh release when GYKI (10-100 microM) was perfused through the hippocampus.Local perfusion with tetrodotoxin (1 microM) decrease the basal release of ACh and prevented the QUIS-induced increase of ACh both in the hippocampus and striatum.Our in vivo neurochemical results indicate that hippocampal and striatal cholinergic systems are regulated by non-NMDA (probably AMPA) glutamatergic receptors located in the hippocampus and striatum.     

Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats

AimsThe effect of exercise training (ET) on vascular responsiveness in diabetes mellitus has been largely well studied. However, limited studies have investigated the effects of ET on functional responses of the corpus cavernosum (CC) in diabetic animals. Therefore, the aim of this study was to investigate whether prior ET prevents the impairment of erectile function in streptozotocin-induced diabetic rats.Main methodsRats were exercised for four weeks prior to the induction of diabetes, and then again for another 4 weeks thereafter. Concentration–response curves to acetylcholine, sodium nitroprusside, Y-27632, BAY 412272 and phenylephrine (PE) were obtained in CC. The excitatory and inhibitory effects of electrical-field stimulation were also evaluated.Key findingsPlasma SOD levels were markedly decreased in the sedentary diabetic group (D-SD) as compared to control sedentary animals (C-SD), approximately 53% (P < 0.05) and this reduction was restored in trained diabetic animals. Physical training restored the impairment of endothelium-dependent and -independent relaxation responses seen in the D-SD group. The potency values for Y-27632 in the CC were significantly reduced in the D-SD group, which was reversed by physical training. The impairment of electrical-field stimulation (EFS)-induced relaxation seen in the D-SD group was restored by physical training. On the other hand, both EFS-induced contractions and concentration–response curves to PE in cavernosal strips were not modified by either diabetes or physical training.SignificancePractice of regular physical exercise may be an important approach in preventing erectile dysfunction associated with diabetes mellitus by re-establishment of the balance between NO production and its inactivation.     

Reconstitution of Cyclin D1-Associated Kinase Activity Drives Terminally Differentiated Cells into the Cell Cycle

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.