Measurement of drug-protein binding by immobilized human serum albumin-HPLC and comparison with ultrafiltration

An HPLC method employing CHIRAL-I (150 mm x 3 mm), 5 microm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to alpha1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.     

Optical microbial biosensor for detection of methyl parathion pesticide using Flavobacterium sp. whole cells adsorbed on glass fiber filters as disposable biocomponent

An optical microbial biosensor was described for the detection of methyl parathion pesticide. Whole cells of Flavobacterium sp. were immobilized by trapping in glass fiber filter and were used as biocomponent along with optic fiber system. Flavobacterium sp. has the organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into detectable product p-nitrophenol. The immobilized microbial biocomponent was disposable, cost-effective and showed high reproducibility and uniformity. The detection of methyl parathion by the use of disposable microbial biocomponent with optical biosensor was simple, single step and direct measurement of very low quantity of the sample. The home made reaction vessel was small and needed only 75 microl of sample. A lower detection limit 0.3 microM methyl parathion was estimated from the linear range (4-80 microM) of calibration plot of organophosphorus hydrolase enzymatic assay. The applicability to synthetic methyl parathion spiked samples was demonstrated.     

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.     

The impact of physical training on neutrophil extracellular traps in young male athletes – a pilot study

Neutrophils are an important component of the innate immune response against various pathogens. However, there is a lack of research concerning the effects of short intensive training on neutrophil functions, especially neutrophil extracellular traps (NET) formation. The study aim was to determine the effects of a 19-day training cycle on innate immunity among young male athletes. Six male ice hockey players (< 20 years old) from the Polish national team were monitored across a five-day training camp and after a return to normal club training. The first blood collection took place before training (T1), the second after the training camp (T2) and the third 14 days later (T3). The counts/concentrations of blood biochemical, immune and endocrine markers were compared across each training period. Creatine kinase activity tended to increase at T2 (546 ± 216 U·L^-1) when compared to T1 (191 ± 111 U·L^-1; p=0.063). Neutrophil extracellular traps formation and neutrophil counts also differed between training periods (p=0.042 and p=0.042, respectively). Neutrophil counts tended to decrease, in contrast to NET formation which tended to rise, at T2 in comparison to T1 (2.51 ± 0.45 vs 3.04 ± 0.47 109·L^-1; 24 ± 13 vs 8 ± 15%, respectively). No significant differences in other leucocyte counts were observed. A short period of intensive training was accompanied by some muscle damage and inflammation, as evidenced by CK and NET up-regulation, whilst neutrophil counts were diminished in the blood. Thus, neutrophils and NET could be involved in muscle damage and local inflammatory processes following intensive physical training in young male athletes.     

Isolation of pyrene degrading <Emphasis Type="Italic">Achromobacter xylooxidans</Emphasis> and characterization of metabolic product

Polycyclic aromatic hydrocarbons (PAHs) are common ubiquitous pollutants existing in nature with high recalcitrance and toxicity. In this study a bacterium capable of aerobic degradation of high molecular weight PAHs (with special reference to pyrene) was isolated by selective enrichment culture technique from oil refinery effluent sludge. The isolate was characterized as Achromobacter xylooxidans by 16S rRNA gene sequence analysis technique. For the first time it is hereby reported a bacterium capable of effectively degrading pyrene (up to 80%), as evident by reverse phase high performance liquid chromatographic analysis (RP-HPLC). After incubation of Achromobacter xylooxidans in minimal salt medium (MSM) containing pyrene, at concentration of 200 mg/L, as sole source of carbon and energy, there was decrease in pyrene concentration concomitant with increase in bacterial cell protein concentration. RP-HPLC analysis revealed that pyrene was degraded into three metabolites viz. I, II and III. The RP-HPLC eluent fraction were collected from 2.5 to 32.0 min by repeated injection of degraded sample, concentrated and analyzed on gas chromatography mass spectroscopy (GC-MS) for metabolite identification. The fraction shows 1-hydroxypyrene, 1-hydroxy-6-methoxypyrene and 1,6dimethoxypyrene as metabolic product of pyrene degradation, on the basis of their m/z values. On contrary to the reported PAH degradation with reference to pyrene by different isolates till date; the efficient degradation, as evident by RP-HPLC, by this isolate holds a promising potential for planning of bioremediation strategies of contaminated sites.     

Satish Chandra Maheshwari (1933–2019)—a brilliant,passionate and an outstanding shining light for all of plant biology

Physiology and Molecular Biology of Plants - We present here a tribute to Satish Chandra Maheshwari (known to many as SCM, or simply Satish), one of the greatest plant biologists of our time. He...     

Exploring Flowering Genes in Isabgol (Plantago ovata Forsk.) Through Transcriptome Analysis

Plant Molecular Biology Reporter - Flowering is one of the major developmental processes that govern the economic yield of crop plants. However, little is known about the molecular mechanisms...