Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

Enhanced glutamate,IP3 and cAMP activity in the cerebral cortex of Unilateral 6-hydroxydopamine induced Parkinson's rats: Effect of 5-HT,GABA and bone marrow cell supplementation

Parkinson's disease is characterized by progressive cell death in the substantia nigra pars compacta, which leads to dopamine depletion in the striatum and indirectly to cortical dysfunction. Increased glutamatergic transmission in the basal ganglia is implicated in the pathophysiology of Parkinson's disease and glutamate receptor mediated excitotoxicity has been suggested to be one of the possible causes of the neuronal degeneration. In the present study, the effects of serotonin, gamma-aminobutyric acid and bone marrow cells infused intranigrally to substantia nigra individually and in combination on unilateral 6-hydroxydopamine induced Parkinson's rat model was analyzed. Scatchard analysis of total glutamate and NMDA receptor binding parameters showed a significant increase in B_max (P < 0.001) in the cerebral cortex of 6-hydroxydopamine infused rat compared to control. Real Time PCR amplification of NMDA2B, mGluR5, bax, and ubiquitin carboxy-terminal hydrolase were up regulated in cerebral cortex of 6-hydroxydopamine infused rats compared to control. Gene expression studies of GLAST, ά-Synuclien and Cyclic AMP response element-binding protein showed a significant (P < 0.001) down regulation in 6-OHDA infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies. Serotonin and GABA along with bone marrow cells in combination showed reversal of glutamate receptors and behaviour abnormality shown in the Parkinson's rat model. The therapeutic significance in Parkinson's disease is of prominence.     

Ratio-dependent parasitism with Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) on egg rafts of Nezara viridula (Linnaeus) (Hemiptera: Pentatomidae): effect of experimental variables and compatibility of 'ratio' and 'Holling' models

Abstract Egg rafts of Nezara viridula were exposed to the parasitoid wasp Trissolcus basalis in experimental arenas to establish the relationship of the rates of attack and parasitism to various combinations of arena size, parasitoid density, host density and parasitoid-to-host ratio. Arena sizes were varied in the ratio 1:9:63, with the largest having a search area of 1.44 m². Parasitoid and host densities were varied over a 27-fold range. The parasitoid-to-host ratios used were 1:1, 3:1 and 6:1. Finding time was related to a constant factor (flight propensity), rather than to the difficulty of finding (density of hosts). Initial attack rates were therefore related only to parasitoid numbers (or density), even at the lower densities and ratios. Parasitism rates (a function of attack rate per host) were thus also strongly related to parasitoid to host ratio, regardless of densities used and arena sizes. Even reducing host density, while keeping time and parasitoid density constant, increased the parasitism rate. A ratio model for parasitism rate was therefore compatible with the data but the more explicit Holling 'disc' equation was also compatible because handling time was sufficiently large to make it sensitive to the ratio of parasitoids to hosts for the densities used. We conclude that the two models would predict different results if the density of host egg rafts was in a range below one per square metre.     

Improvement of psoriasis after tonsillectomy is associated with a decrease in the frequency of circulating T cells that recognize streptococcal determinants and homologous skin determinants

Exacerbation of chronic psoriasis can be associated with streptococcal throat infections, and T cells that respond to peptide sequences common to streptococcal M proteins and skin keratins have been detected in patients' blood. To our knowledge, we have conducted the first blinded, prospective study to assess the impact of tonsillectomy on psoriasis. Twenty-nine patients with chronic psoriasis and history of exacerbation after sore throat were randomly assigned to tonsillectomy (n = 15) or control (n = 14) groups and monitored for 2 y clinically and by enumeration of circulating skin homing T cells that respond to short homologous M protein or keratin peptides. Thirteen patients (86%) showed sustained improvement after tonsillectomy ranging from 30 to 90% reduction in disease severity. Furthermore, there was a close correlation between the degree of clinical improvement in individual patients and reduction in the frequency of peptide-reactive skin-homing T cells in their circulation. No corresponding clinical or immunologic changes were observed among the controls. These findings indicate that tonsillectomy may have a beneficial effect on chronic psoriasis because the palatine tonsils generate effector T cells that recognize keratin determinants in the skin.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

Biochemical predictors for Sars-Cov-2 severity

It is of interest to assess the inflammatory marker profile in SARS-CoV-2 patients and to correlate the levels of systemic inflammatory biomarkers such as neutrophil-to-lymphocyte ratio (NLR), C-Reactive Protein CRP, Ferritin, Creatine kinase (CK), Lactate dehydrogenase (LDH) and liver function analytes total serum proteins, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with the severity of SARS-CoV-2 infections. A total of 1000 COVID-19 positive patient''s data were collected. Laboratory assessments consisted of NLR (neutrophil-lymphocyte ratio) by cell counter, C Reactive Protein (CRP) by immunoturbidimetry, Ferritin by electrochemiluminescence (ECLIA) and Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Total Protein and Albumin by spectrophotometry. The mean plasma CRP levels, NLR, ferritin, CK and LDH levels were higher in severe cases than in non-severe cases, and the difference was statistically significant (p<0.05). All liver function tests such as the total and direct bilirubin, AST, ALT, ALP, total protein and albumin were higher in severe patients than non-severe patients and the difference was statistically significant (p<0.05). Data indicate that NLR, CRP, Ferritin, CK, LDH and liver function analytes have a crucial role as prognostic markers for SARS-CoV-2 infections and hence should be routinely recommended for risk assessment and stratification of the patients to reduce the associated morbidity and mortality.