Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation

Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) alpha and PDGFRbeta. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRbeta; this involves the Cbl TKB domain binding to PDGFRbeta phosphotyrosine 1021, a known phospholipase C (PLC) gamma1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRbeta impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLCgamma1 association with PDGFRbeta and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRbeta involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.     

Physiological responses during continuous work in hot dry and hot humid environments in Indians

Studies have been conducted on six young healthy heat acclimatised Indians to determine the physiological changes in prolonged continuous work in thermally neutral and in hot dry and hot humid environments. Physiological responses in maximal efforts i.e. Vo₂ max, V_E max and Cf max were noted. In addition, duration in continuous work at three sub-maximal rate of work in three simulated environments were also noted. Physiological responses like Vo₂, V_E and Cf were noted every 15 minutes of work. Besides these responses, rectal temperature (Tre), mean skin temperature (T_s) and mean sweat rate were also recorded during continuous work.Results indicated a significant decrease in maximum oxygen uptake capacity (Vo₂ max) in heat with no change in maximum exercise ventilation (V_E max) and maximum cardiac frequency. However, the fall in Vo₂ max was more severe in the hot humid environment than in the hot dry climate. Cardiac frequency at fixed oxygen consumption of 1.0, 1.5 and 2.0 l/min was distinctly higher in the hot humid environment than in the hot dry and comfortable temperature. The duration in continuous physical effort in various grades of activities decreased in hot dry environment from that in the-comfortable climate and further decreased significantly in hot humid environment. The highest rate of sweating was observed during work in humid heat. The mean skin temperature (T_s) showed a fall in all the three rates of work in comfortable and hot dry conditions whereas in hot humid environment it showed a linear rise during the progress of work. The rectal temperature on the other hand maintained a near steady state while working at 65 and 82 watts in comfortable and hot dry environments but kept on rising during work in hot humid environment. At the highest work rate of 98 watts, the rectal temperature showed a steady increase even in the hot dry condition. It was thus concluded from the study that a hot humid climate imposes more constraints on the thermoregulatory system during work than in the hot dry condition because of less effective heat dissipation so resulting in reduced tolerance to work.     

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients

Tuberculous meningitis (TBM) is a serious complication of tuberculosis that affects the central nervous system. Present methods to diagnose TBM are not suitable for early diagnosis. Molecular markers and sensitive methods to identify them in the early stage of infection of TBM are critically needed for efficient management. We have done the proteomic analysis of TBM cerebrospinal fluid (n=20) with 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 11 human proteins and 8 mycobacterial proteins with changed expression levels in comparison to controls. Arachidonate 5-lipoxygenase and glial fibrillary acidic protein, two of the identified proteins, were validated with western blot technique on a larger set of disease and control samples (n=40). These two proteins were also analyzed in fungal meningitis samples. We suggest that arachidonate 5-lipoxygenase can be considered for validation as a potential marker for diagnosis of TBM.     

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.Key words: BMI1, βTrCP, polycomb group proteins, senescence, breast cancer     

Sirtuin1: A Promising Serum Protein Marker for Early Detection of Alzheimer’s Disease

Sirtuin (SIRT) pathway has a crucial role in Alzheimer’s disease (AD). The present study evaluated the alterations in serum sirtuin1 (SIRT1) concentration in healthy individuals (young and old) and patients with AD and mild cognitive impairment (MCI). Blood samples were collected from 40 AD and 9 MCI patients as cases and 22 young healthy adults and 22 healthy elderly individuals as controls. Serum SIRT1 was estimated by Surface Plasmon Resonance (SPR), Western Blot and Enzyme Linked Immunosorbent Assay (ELISA). A significant (p<0.0001) decline in SIRT1 concentration was observed in patients with AD (2.27±0.46 ng/µl) and MCI (3.64±0.15 ng/µl) compared to healthy elderly individuals (4.82±0.4 ng/µl). The serum SIRT1 concentration in healthy elderly was also significantly lower (p<0.0001) compared to young healthy controls (8.16±0.87 ng/µl). This study, first of its kind, has demonstrated, decline in serum concentration of SIRT1 in healthy individuals as they age. In patients with AD and MCI the decline was even more pronounced, which provides an opportunity to develop this protein as a predictive marker of AD in early stages with suitable cut off values.     

Synthesis and spectral investigations of Cu(II) ion‐doped NaCaAlPO4F3 phosphor

Cu(II) ion‐doped NaCaAlPO₄F₃ phosphor has been synthesized using a solid state reaction method. The prepared sample is characterized by powder X‐ray diffraction, scanning electron microscope, optical absorption, electron paramagnetic resonance photoluminescence and Fourier transform infrared spectroscopy techniques. The crystallite size evaluated from x‐ray diffraction data is in nanometers. Scanning electron microscopy micrographs showed the presence of several irregular shaped particles. From optical absorption and electron paramagnetic resonance spectral data the doped Cu(II) ions are ascribed to distorted octahedral site symmetry. The synthesized phosphor exhibits emission bands in ultraviolet, blue and green regions under the excitation wavelength of 335 nm. The CIE chromaticity coordinates (x = 0.159, y = 0.204) also calculated for the prepared sample from the emission spectrum. The Fourier transform infrared spectroscopy spectrum revealed the characteristic vibrational bands of the prepared phosphor material. Copyright © 2014 John Wiley & Sons, Ltd.     

A rain forest dusk chorus: cacophony or sounds of silence?

A rain forest dusk chorus consists of a large number of individuals of acoustically communicating species signaling at the same time. How different species achieve effective intra-specific communication in this complex and noisy acoustic environment is not well understood. In this study we examined acoustic masking interference in an assemblage of rain forest crickets and katydids. We used signal structures and spacing of signalers to estimate temporal, spectral and active space overlap between species. We then examined these overlaps for evidence of strategies of masking avoidance in the assemblage: we asked whether species whose signals have high temporal or spectral overlap avoid calling together. Whereas we found evidence that species with high temporal overlap may avoid calling together, there was no relation between spectral overlap and calling activity. There was also no correlation between the spectral and temporal overlaps of the signals of different species. In addition, we found little evidence that species calling in the understorey actively use spacing to minimize acoustic overlap. Increasing call intensity and tuning receivers however emerged as powerful strategies to minimize acoustic overlap. Effective acoustic overlaps were on average close to zero for most individuals in natural, multispecies choruses, even in the absence of behavioral avoidance mechanisms such as inhibition of calling or active spacing. Thus, call temporal structure, intensity and frequency together provide sufficient parameter space for several species to call together yet communicate effectively with little interference in the apparent cacophony of a rain forest dusk chorus.     

Influence of Matrix Metalloproteinase-1 Gene −1607 (1G/2G) (rs1799750) Promoter Polymorphism on Circulating Levels of MMP-1 in Chronic Pancreatitis

This study investigated the role of ?1607 (1G/2G) (rs1799750) polymorphism of the MMP-1 gene in chronic pancreatitis. We genotyped 100 patients with chronic pancreatitis and 100 control subjects using tetra-primer ARMS-PCR followed by agarose gel electrophoresis. Serum levels of MMP-1 were determined by Elisa. Statistical analysis was applied to test the significance of the results. The genotypic and allelic distribution varied significantly between the disease group and the control subjects [OD = 1.981 (1.236–3.181), p = 0.004]. MMP-1 levels were higher in subjects homozygous for the 2G allele than in subjects with the 1G allele. The present study revealed a significant association of the MMP-1 ?1607 1G/2G (rs1799750) gene promoter polymorphism with chronic pancreatitis, and it can be considered a biological marker in the etiology of chronic pancreatitis.     

Effect of Anacyclus pyrethrum on Pentylenetetrazole-Induced Kindling, Spatial Memory, Oxidative Stress and Rho-Kinase II Expression in Mice

Anacyclus pyrethrum (A. pyrethrum) has been reported to exhibit anticonvulsant activity. In the present study, the effect of hydro-alcoholic extract of A. pyrethrum root (HEAP) on pentylenetetrazole (PTZ) induced kindling, spatial memory, oxidative stress and rho kinase (ROCK II) was assessed. Male albino mice (25–30 g) were used in the study. PTZ (35 mg/kg, i.p. on alternate days) was injected to induce kindling and PTZ (70 mg/kg, i.p) challenge was given 7 days post-kindling. HEAP was administered orally daily in the doses of 100, 250 and 500 mg/kg along with PTZ injections during the kindling process and continued till PTZ challenge post kindling. Spatial memory was assessed using Morris water maze test. Oxidative stress parameters [malondialdehyde (MDA) and reduced glutathione (GSH)] and ROCK II expression were estimated in whole brain at the end of the study. Pre-treatment with HEAP (250 and 500 mg/kg) showed significant increase in the myoclonic jerk latency and delay in the development of kindling. A significant decrease in mortality was observed at higher doses of HEAP (250 and 500 mg/kg). Pre-treatment with HEAP significantly increased the number of platform crossings and decreased the escape latency, as opposed to the PTZ group, thus showing protection against memory deficit. HEAP pre-treatment also attenuated the oxidative stress induced by PTZ kindling. PTZ induced kindling increased the ROCK II expression whereas, HEAP pre-treatment attenuated the increase in ROCK II expression. To conclude, HEAP pre-treatment showed antiepileptic effect and also showed protection against cognitive impairment by decreasing oxidative stress and ROCK II expression in PTZ kindled mice.