The effect of time of day and exercise on platelet functions and platelet–neutrophil aggregates in healthy male subjects

Platelet activation state changes by exercise. The effect of exercise time on platelet activation state and formation of platelet–neutrophil aggregates are not known yet. In this study the effect of exercise and time of day were examined on platelet activity with platelet–neutrophil aggregates. Ten moderately active males aged 27± 1.63 (mean±S.D.) years completed sub-maximal (70% VO_2max) exercise trials for 30 min. Blood pressure (BP) was recorded. Venous blood samples were obtained at rest, immediately post-exercise and after 30 min of recovery. Whole blood was analysed for haematocrit (Hct), haemoglobin (Hb), platelet count (PC), mean platelet count (MPV) and platelet aggregation (PA). Platelet–neutrophil aggregates and beta-thromboglobulin (β-TG) levels were assayed. Platelet count showed significant increase after morning exercise ((236± 32)×10⁹ l⁻¹ versus (202± 34)×10⁹ l⁻¹ baseline, p < 0.05). Exercise resulted in significantly lower MPV after the evening exercise (9.16± 0.5 fl versus 9.65± 0.36 fl, p < 0.05). Platelet aggregation by adenosine diphosphate (ADP) decreased after morning exercise and the recovery aggregation levels were significantly different at two different times of the day (68± 20% a.m. versus 80± 12% p.m., p < 0.05). It was also showed that platelet–neutrophil aggregates increased significantly from baseline after both exercises. Exercise-induced platelet–neutrophil aggregates were higher in the evening (10.7± 1.3% p.m. versus 6.4± 1.8% a.m., p < 0.0001). It is therefore concluded that besides platelet–platelet aggregation, exercise can cause platelet– neutrophil aggregates. In addition, time of day has an effect on platelet activation related events. Circadian variations of physiological parameters may have an effect on thrombus formation by platelet activation. (Mol Cell Biochem xxx: 119–124, 2005)     

The effect of glutamine on radiation-induced organ damage

Radiation enteritis is a significant clinical problem in patients receiving ionizing radiation directed to the abdomen or pelvis. Although radiation is aimed to be directed against the malignant tissue, adjacent healthy tissues are also affected. The small intestine is the most sensitive organ to radiation. The present study was undertaken to investigate the possible protective effect of glutamine against radiation-induced intestinal, hepatic and pancreatic toxicity. Rats received 1 g/kg/day glutamine for seven days before irradiation and continued for three days after irradiation until sacrifice. Then intestinal, pancreatic and hepatic myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels and caspase-3 activities of the sacrificed rats were measured. Irradiation significantly increased the intestinal and pancreatic MPO and caspase-3 activities and MDA levels in comparison to sham group. Glutamine treatment significantly decreased this elevation. Histopathological examination revealed that the intestinal mucosal structure was preserved and pancreatic inflammation decreased in the glutamine treated group. In irradiation group, NF-kappaB over expression was detected. There was no significant difference in histopathological and biochemical examinations of the liver between the groups. In conclusion, glutamine has beneficial effects on intestinal and pancreatic damage in abdominal irradiation through the inflammatory process and apoptosis.     

A molecular defect in intracellular lipid signaling in human neutrophils in localized aggressive periodontal tissue damage

Host defense mechanisms are impaired in patients with congenital neutrophil (polymorphonuclear neutrophils (PMN)) defects. Impaired PMN chemotaxis is observed in localized aggressive periodontitis (LAP), a familial disorder characterized by destruction of the supporting structures of dentition. In the present studies, we sought evidence for molecular events underlying this aberrant human PMN phenotype. To this end, PMN transendothelial migration and superoxide anion generation were assessed with LAP patients and asymptomatic family members, as well as patients with other chronic mucosal inflammation. PMN from LAP patients showed decreased transmigration across vascular endothelial monolayers (18 +/- 12% of control, n = 4) and increased superoxide anion generation (358 +/- 37%, p = 0.003). Gene expression was analyzed using oligonucleotide microarrays and fluorescence-based kinetic PCR. cDNA microarray and kinetic-PCR analysis revealed diminished RNA expression of leukocyte-type diacylglycerol (DAG) kinase alpha in PMN from LAP patients (4.6 +/- 1.7 relative units, n = 6, p = 0.007) compared with asymptomatic individuals (51 +/- 27 relative units, n = 7). DAG kinase activity was monitored by DAG phosphorylation and individual DAG molecular species were quantified using liquid chromatography and tandem mass spectrometry-based lipidomics. DAG kinase activity was also significantly decreased (73 +/- 2%, p = 0.007) and correlated with increased accumulation of 1,2-diacyl-sn-3-glycerol substrates (p = 0.01). These results implicate defects in both PMN transendothelial migration and PMN DAG kinase alpha signaling as disordered functions in LAP. Moreover, they identify a potential molecular lesion in PMN signal transduction that may account for their aberrant responses and tissue destruction in this disease.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Age and growth of the hollowsnout grenadier, Caelorinchus caelorhincus (Risso, 1810), in the Aegean Sea

Age and growth of the hollowsnout grenadier was studied based on samples collected seasonally in the Agean Sea between March 2003 and January 2004. Total length ranged from 9.0 to 21.6 cm. Length–weight relationship was described as a = 0.0032, and b = 3.008. Age determinations based on otolith readings were between 1 and 10 years. The von Bertalanffy growth curve fitted to observed lengths‐at‐age provided parameters of L_∞ = 24.9 cm, k = 0.115 and t_o = ?3.494.     

Molecular dynamics simulations of apo,holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

The pyridoxal 5‐phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ‐aminobutyric acid aminotransferase (GABA‐AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA‐AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA‐AT. This is a consequence of a pK_a shift triggered by a strong charge–charge interaction with an ionic “diad” formed by Asp298 and His190 that would help the activation of the first half‐reaction of the catalytic mechanism in GABA‐AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π–π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half‐reaction in GABA‐AT: the regeneration of PLP‐bound GABA‐AT (i.e., the holoenzyme). Proteins 2016; 84:875–891. © 2016 Wiley Periodicals, Inc.     

Computational design of Phe-Tyr dipeptide and preparation,characterization, cytotoxicity studies of Phe-Tyr dipeptide loaded PLGA nanoparticles for the treatment of hypertension

Phe-Tyr dipeptide which was investigated in Wakame food with greatest ACE-inhibitory activity is used as a pharmaceutical drug for the treatment of hypertension, cardiovascular diseases, and diabetic nephropathy. To improve the bioavailability of Phe-Tyr, a delivery system based on poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with Phe-Tyr (Phe-Tyr-PLGA NPs) for treating hypertension and cardiovascular diseases was prepared in this study. In the experiments, poly(lactic-co-glycolic acid) (PLGA) and Phe-Tyr dipeptide-loaded PLGA nanoparticles were prepared using the double emulsion (w/o/w) method. The characterizations of the nanoparticles were performed with a UV–vis spectrometer, the Zeta-sizer system, and FTIR spectrometer. The optimum size of the Phe-Tyr dipeptide-loaded PLGA nanoparticle was obtained with a 213.8 nm average particle size, and a 0.061 polydispersity index, ?19.5 mV zeta potential, 34% of loaded and 90.09% of encapsulation efficiency. From TEM analysis, it was clearly seen that the dipeptide loaded nanoparticles had the spherical and non-aggregated morphology and Phe-Tyr dipeptide loaded-PLGA nanoparticles were obtained successfully. Cell toxicity of nanoparticles at different concentrations was assayed with XTT methods on L929 fibroblast cells. This study determined that the nanoparticles have low toxicity at lower concentration and toxicity augmented with increasing concentration of dipeptide. To analyze the effect of solvents on structure of Phe-Tyr, Molecular dynamics simulation was performed with GROMACS program and molecular orbital calculations were carried out to obtain structural and electronic properties of dipeptide. Moreover, molecular docking calculations were also employed to model and predict protein–drug interactions.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.