Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822).

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28°C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.

Rictor regulates cell migration by suppressing RhoGDI2.

Rictor and its binding partner Sin1 are indispensable components of mTORC2 (mammalian target of rapamycin complex 2). The mTORC2 signaling complex functions as the regulatory kinase of the distinct members of AGC kinase family known to regulate cell proliferation and survival. In the early chemotaxis studies in Dictyostelium, the rictor's ortholog has been identified as a regulator of cell migration. How rictor regulates cell migration is poorly characterized. Here we show that rictor regulates cell migration by controlling a potent inhibitor of Rho proteins known as the Rho-GDP dissociation inhibitor 2 (RhoGDI2). On the basis of on our proteomics study we identified that the rictor-dependent deficiency in cell migration is caused by upregulation of RhoGDI2 leading to a low activity of Rac and Cdc42. We found that a suppression of RhoGDI2 by rictor is not related to the Sin1 or raptor function that excludes a role of mTORC2 or mTORC1 in regulation of RhoGDI2. Our study reveals that rictor by suppressing RhoGDI2 promotes activity of the Rho proteins and cell migration.

Production and characterization of a monoclonal antibody against putative T lymphocytes of Catla catla.

Catla catla is the fastest growing Indian major carp and one of the major aquaculture species in South Asia. A monoclonal antibody (MAb) designated B8 MAb was produced against nylon wool-enriched thymus mononuclear cells of C. catla. This MAb did not show reactivity with macrophage and epithelial cell lines derived from catla thymus in cellular ELISA. In flow cytometric analysis of gated lymphocytes, the percentage of B8 positive (B8+) cells in thymus (n = 10, 500-600 g) was determined to be 77.7 %. Similarly, the percentage of B8+ cells in kidney, spleen and blood (n = 5) was 15.08, 1.1 and 32.17 %, respectively. Western blotting of reduced membrane proteins showed that B8 MAb reacted with a polypeptide having a molecular weight of 168.2 kDa. In indirect immunoperoxidase test, B8+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio. B8 reactive cells were densely packed in central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. B8 MAb also reacted with a significant population of lymphocytes in blood smears. Considering the economic importance of C. catla, this MAb should be a useful tool for studying immune response of this fish species.

PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma.

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patient's abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

Peptidyl-prolyl cis/trans isomerase Pin1 is critical for the regulation of PKB/Akt stability and activation phosphorylation.

The serine/threonine protein kinase B (PKB, also known as Akt) plays a pivotal role in diverse cellular functions. Elevated expression of activated Akt has been detected in a wide variety of human cancers; however, the mechanism of Akt protein stability regulation remains unclear. In this study, we showed a strong correlation between the expression levels of an oncogenic peptidyl-prolyl cis/trans isomerase Pin1 and levels of Akt phosphorylation at S473 in multiple cancer types (P<0.0001). Akt-pS473 status combined with Pin1 expression levels predicted a poorer prognosis than did either one alone in patients with breast cancer (P=0.0052). We further showed that Pin1 regulated Akt stability and phosphorylation on S473 through the phosphorylated Thr-Pro motifs of Akt. These motifs are conserved evolutionary and are required for the maintenance of Akt stability and its interaction with Pin1. In addition, repressing Pin1 expression through either homologue Pin1 knockout or small interfering RNA-mediated knockingdown compromised its ability to protect Akt from degradation. Our results show how Akt protein stability is regulated by the peptidyl-prolyl cis/trans isomerase Pin1 and highlight the importance of this oncogenic network in human disease pathogenesis.

Deep vein thrombosis in acute spinal cord injury.

STUDY DESIGN: Randomized study. OBJECTIVES: To find out the incidence of deep vein thrombosis (DVT) in acute spinal cord injury (SCI) patients with and without therapeutic prophylaxis. SETTING: Patients admitted in the department of Physical Medicine & Rehabilitation, SMS Medical College, Jaipur, India. METHODS: All 297 patients received physical therapy measures and were randomly divided into two groups. 166 patients received prophylactic heparin, whereas 131 patients did not. RESULTS: A total of three cases (1.8%) in study group and four cases (3%) in control group developed DVT. This difference was statistically insignificant (P>0.05). CONCLUSION: Incidence of DVT in SCI is low in our study.

Medical certification in respect of alleged torture victims.

Torture is a global problem and affects a large number of people worldwide. The opinion of doctors in certifying various types of physical, and psychological injuries being caused accidentally or resulting from attempted suicide or by torture is very significant. A number of times, discrepancies between complaints and physical findings are noted. Meticulous examination is desirable in order to prove guilt or innocence.

SEM surface structure of the adhesive organ of the hillstream fish Glyptothorax pectinopterus (Teleostei: Sisoridae) from the Garhwal Hills.

Glyptothorax pectinopterus has a well defined bilateral adhesive organ situated posteriorly to the mouth, between the opercular opening and the base of the pectoral fin. It narrows at its anterior and posterior end and is characterized by thin longitudinal ridges and grooves running almost parallel to the main body axis, but with a slight inclination posteriorly. The ridges all seem to converge towards the posteriorly localized middle pad. In conformity with the hillstream type of habitat, the ridges are surmounted by long, keratinized epidermal spines with hooked, curved or sucker-like tips. The hooked spines, in adhesion to the substratum, attach themselves firmly to organic material, while the sucker-tipped spines reinforce adhesion by generating vacua at innumerable scattered points. The effect of mechanical abrasion of the spines is minimized by a glandular secretion from the middle pad.

The adhesion of thin carbon films to metallic substrates.

As part of the development of carbon-coated prosthetic devices, the adhesion of thin carbon films to metallic substrates has been studied. The bond strength of carbon films about 5000 A thick on Ti-6A1-4V and stainless steel was measured in a pull test and found to be greater than 4700 psi. Auger electron spectroscopy showed a reactive film/substrate interface. The ultimate bond strength was found to be dependent on the substrate and the deposition parameters.