Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

Scanning electron microscopy of the heart of the climbing perch

The air-breathing climbing perch Anabas testudineus has two ventral aortas, one directs blood through well developed anterior gill arches into the suprabranchial chambers and back to the heart and the other sends blood through rudimentary shunt-like posterior arches and onto the systemic circulation. The sinus venosus is a thin-walled structure and lacks myocardial trabeculae. The atrium is similar to that of other teleosts and it is traversed by numerous myocardial trabeculae. There are no sinoatrial valves, whereas the atrioventricular aperture is guarded by one pair of large wing-shaped and two small cap-shaped valves. The ventricle is composed only of spongy myocardium and has numerous branching lacunae extending to the epicardium. The thick-walled bulbus arteriosus is lined with longitudinal ridges and this and the ventricular trabeculae may minimize mixing of respiratory and systemic flow while blood is passing through the heart. However, with the exception of the absence of sinoatrial valves and the ridged bulbar lumen, the heart of the climbing perch is essentially similar to that of most other non air-breathing teleosts.     

Polymorphisms in CaSR and CLDN14 Genes Associated with Increased Risk of Kidney Stone Disease in Patients from the Eastern Part of India

Kidney stone disease (KSD) is a major clinical problem imposing a large burden for both healthcare and economy globally. In India, the prevalence of kidney stone disease is rapidly increasing. This study aimed to evaluate the association between genetic defects in vitamin D receptor (VDR), calcium sensing receptor (CaSR) and claudin 14 (CLDN14) genes and kidney stone disease in patients from eastern India. We enrolled 200 consecutive kidney stone patients (age 18–60 years) (cases) and their corresponding sex and age matched 200 normal individuals (controls). To identify genetic variants responsible for KSD, we performed sequence analysis of VDR, CaSR and CLDN14 genes. Four non-synonymous (rs1801725, rs1042636, rs1801726 and rs2228570), one synonymous (rs219780) and three intronic single nucleotide polymorphisms (SNPs) (rs731236, rs219777 and rs219778) were identified. Genotype and allele frequency analysis of these SNPs revealed that, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) of CaSR gene and rs219778, rs219780 (Thr229Thr) of CLDN14 gene were significantly associated with KSD. Serum calcium levels were significantly higher in subjects carrying 986Ser allele and calcium excretion was higher in subjects bearing 990Gly allele. In conclusion, rs1801725, rs1042636, rs219778 and rs219780 SNPs were associated with kidney stone risk in patients from the eastern part of India.     

Comparative Nuclear Proteomics Analysis Provides Insight into the Mechanism of Signaling and Immune Response to Blast Disease Caused by Magnaporthe oryzae in Rice

Modulation of plant immune system by extrinsic/intrinsic factors and host‐specific determinants fine‐tunes cellular components involving multiple organelles, particularly nucleus to mount resistance against pathogen attack. Rice blast, caused by hemibiotrophic fungus Magnaporthe oryzae, is one of the most devastating diseases that adversely affect rice productivity. However, the role of nuclear proteins and their regulation in response to M. oryzae remains unknown. Here, the nucleus‐associated immune pathways in blast‐resistant rice genotype are elucidated. Temporal analysis of nuclear proteome is carried out using 2‐DE coupled MS/MS analysis. A total of 140 immune responsive proteins are identified associated with nuclear reorganization, cell division, energy production/deprivation, signaling, and gene regulation. The proteome data are interrogated using correlation network analysis that identified significant functional modules pointing toward immune‐related coinciding processes through a common mechanism of remodeling and homeostasis. Novel clues regarding blast resistance include nucleus‐associated redox homeostasis and glycolytic enzyme–mediated chromatin organization which manipulates cell division and immunity. Taken together, the study herein provides evidence that the coordination of nuclear function and reprogramming of host translational machinery regulate resistance mechanism against blast disease.     

Cross-talk between macrophages,smooth muscle cells,and endothelial cells in response to cigarette smoke: the effects on MMP2 and 9

The aim of the present study was to analyze the expression of sex-determining region Y-related high mobility group box 4 (SOX4) in non-small cell lung cancer (NSCLC) and its correlation with clinicopathologic characteristics, including the survival of NSCLC patients. To observe initially the expression status of SOX4 in lung squamous cell carcinoma and adenocarcinoma at gene expression omnibus. The expression of SOX4 mRNA and protein was examined in NSCLC tissues and normal lung tissues through real-time PCR and immunohistochemistry. Meanwhile, the relationship of SOX4 expression levels with clinical characteristics of 168 NSCLC patients was analyzed by immunohistochemistry. Univariate and multivariate analyses were performed to determine the association between SOX4 expression and prognosis of NSCLC patients. In our results, SOX4 expression was increased in NSCLC tissues compared with paired normal lung tissues in microarray data (GSE3268). SOX4 mRNA and protein expression were markedly higher in NSCLC tissues than in normal lung tissues (P = 0.001 and P = 0.001, respectively). Using immunohistochemistry, high levels of SOX4 protein were positively correlated with status of differentiated degree (high vs. middle, P = 0.004; high vs. low, P < 0.001), clinical stage (I–II vs. III–IV, P < 0.001), T classification (T1–T2 vs. T3–T4, P = 0.004), N classification (N0–N1 vs. N2–N3, P = 0.002), and M classification (M0 vs. M1, P = 0.011) in NSCLC. Moreover, the higher level of SOX4 expression was markedly correlated with poor overall survival in NSCLC patients (P < 0.001). Multivariate analysis suggested that increased SOX4 expression was a poor independent prognostic predictor for NSCLC patients (P = 0.002). In conclusion, SOX4 plays an important role on NSCLC progression and prognosis and may serve as a convictive prognostic biomarker for NSCLC patients.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.