Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging,Cell Injury Measurements,and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.     

Lymphatic Endothelial Differentiation in Pulmonary Lymphangioleiomyomatosis Cells

Pulmonary lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm affecting almost exclusively women of childbearing age. LAM belongs to the family of perivascular epithelioid cell tumors, characterized by spindle and epithelioid cells with smooth muscle and melanocytic differentiation. LAM cells infiltrate the lungs, producing multiple, bilateral lesions rich in lymphatic channels and forming cysts, leading to respiratory insufficiency. Here we used antibodies against four lymphatic endothelial markers—podoplanin (detected by D2-40), prospero homeobox 1 (PROX1), vascular endothelial growth factor receptor 3 (VEGFR-3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)—to determine whether LAM cells show lymphatic differentiation. Twelve of 12 diagnostic biopsy specimens (early-stage LAM) and 19 of 19 explants (late-stage LAM) showed immunopositivity for D2-40 in most neoplastic cells. PROX1, VEGFR-3, and LYVE1 immunoreactivity varied from scarce in the early stage to abundant in the late stage. Lymphatic endothelial, smooth muscle, and melanocytic markers were partially co-localized. These findings indicate that lymphatic endothelial differentiation is a feature of LAM and provide evidence of a previously unidentified third lineage of differentiation in this neoplasm. This study has implications for the histological diagnosis of LAM, the origin of the neoplastic cells, and potential future treatment with drugs targeting lymphangiogenesis.     

Non-Random Variability in Functional Composition of Coral Reef Fish Communities along an Environmental Gradient

Changes in the coral reef complex can affect predator-prey relationships, resource availability and niche utilisation in the associated fish community, which may be reflected in decreased stability of the functional traits present in a community. This is because particular traits may be favoured by a changing environment, or by habitat degradation. Furthermore, other traits can be selected against because degradation can relax the association between fishes and benthic habitat. We characterised six important ecological traits for fish species occurring at seven sites across a disturbed coral reef archipelago in Indonesia, where reefs have been exposed to eutrophication and destructive fishing practices for decades. Functional diversity was assessed using two complementary indices (FRic and RaoQ) and correlated to important environmental factors (live coral cover and rugosity, representing local reef health, and distance from shore, representing a cross-shelf environmental gradient). Indices were examined for both a change in their mean, as well as temporal (short-term; hours) and spatial (cross-shelf) variability, to assess whether fish-habitat association became relaxed along with habitat degradation. Furthermore, variability in individual traits was examined to identify the traits that are most affected by habitat change. Increases in the general reef health indicators, live coral cover and rugosity (correlated with distance from the mainland), were associated with decreases in the variability of functional diversity and with community-level changes in the abundance of several traits (notably home range size, maximum length, microalgae, detritus and small invertebrate feeding and reproductive turnover). A decrease in coral cover increased variability of RaoQ while rugosity and distance both inversely affected variability of FRic; however, averages for these indices did not reveal patterns associated with the environment. These results suggest that increased degradation of coral reefs is associated with increased variability in fish community functional composition resulting from selective impacts on specific traits, thereby affecting the functional response of these communities to increasing perturbations.     

Comprehensive <Superscript>1</Superscript>H NMR metabolic profiling of body fluids for differentiation of meningitis in adults

Introduction

Meningitis, a morbidly infectious central nervous system pathology is accompanied by acute inflammation of the meninges, causing raised intracranial pressure linked with serious neurological sequelae.

Objective

To observe the variation in the metabolic profile, that may occur in serum and urine along with CSF in adults using ¹H NMR spectroscopy, with an attempt of appropriate and timely treatment regimen.

Methods

The ¹H NMR-based metabolomics has been performed in 115 adult subjects for differentiating bacterial meningitis (BM) and tubercular meningitis (TBM).

Results

The discriminant function analysis (DFA) of the three bio-fluids collectively identified 3-hydroxyisovalerate, lactate, glucose, formate, valine, alanine, ketonic bodies, malonate and choline containing compounds (choline and GPC) as significant metabolites among cases versus control group. The differentiation of bacterial meningitis and tuberculous meningitis (BM vs. TBM) can be done on the basis of identification of 3-hydroxyisovalerate, isobutyrate and formate in case of CSF (with a correct classification of 78 %), alanine in serum (correct classification 60 %), valine and acetone in case of urine (correct classification 89.1 %). The NMR spectral bins based orthogonal signal correction principal component analysis score plots of significant metabolites obtained from DFA also provided group classification among cases versus control group in CSF, serum and urine samples. The variable importance in projection scores also identified similar significant metabolites as obtained from DFA, collectively in CSF, serum and urine samples, responsible for differentiation of meningitis.

Conclusion

The CSF contained metabolites which are formed during infection and inflammation, and these were also found in significant quantity in serum and urine samples.

     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Conformations of hydrophobic peptides in trifluoroethanol, water and in solid state: a circular dichroism and Fourier Transform Infrared study

The conformations of peptides corresponding to KLLIALVLCFLPLAALG have been examined in trifluoroethanol (TFE), aqueous medium by circular dichroism spectroscopy and in the solid state by Fourier Transform Infra Red Spectroscopy (FTIR). The 17-residue parent peptide and peptides corresponding to shorter segments LVLCFLPLAALG and CFLPLAALG showed preference for helical conformation in TFE. Even the shorter hydrophobic peptides corresponding to KLLIA and LVL showed propensity for beta-turn conformations in TFE. However, peptides corresponding to the relatively polar segment FLPLAALG were unordered in TFE. In water, peptides that showed ordered conformation in TFE preferred beta-conformation. In solid-state, FTIR spectra indicated that the hydrophobic peptides adopt beta-structures with extensive hydrogen bonded network in the solid-state. The hydrophobic core segment thus appears to dictate the conformational propensity of the peptide.     

RhoA-Rho kinase mediates synergistic ET-1 and phenylephrine contraction of rat corpus cavernosum

Maintenance of the detumescent state of the penis is believed to involve the actions of several vasoconstrictors. However, our mechanistic understanding of any synergistic vasoconstrictor influences is extremely limited. We tested the hypothesis that a vasoconstrictor combination of endothelin (ET-1) and phenylephrine (PE) augments the constrictor responses in rat corporal cavernosal tissues by a mechanism involving the RhoA-Rho kinase pathway. Independently, ET-1 (1 nM-30 microM) and PE (100 nM-100 microM) both caused dose-dependent contractions of isolated rat cavernosal tissues. In combination, ET-1 (30 nM) augmented the contractile effect of PE and shifted the calculated EC50 for PE (90 +/- 12 to 45 +/- 5 microM). The active stress generated by cavernosal strips during the ET-1 + PE combined stimulation (4.9 +/- 0.2 mN/mm2) was greater than the combined stress generated with ET-1 (0.4 +/- 0.1 mN/mm2) or PE (3.3 +/- 0.2 mN/mm2) stimulations alone. Blockade of ETA receptors (30 nM; A-127722) reversed the augmented stress generation and the Rho-kinase inhibitor Y-27632 differentially and dose-dependently relaxed the tissue. The combined constrictor effect was associated with a fourfold increase of RhoA in the membrane faction of the tissue homogenates. We conclude that the ET-1 + PE combination potentiate vasoconstriction through mutual activation of the RhoA-Rho kinase pathway. The interactions of these agonists likely play important roles in the maintenance of the flaccid state and contribute to some forms of erectile dysfunction.     

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.     

Oxidative injury due to chronic nitric oxide synthase inhibition in rat: effect of regular exercise on the heart

Many individuals with cardiac diseases undergo periodic physical conditioning with or without medication. Therefore, this study investigated the interaction of physical training and chronic nitric oxide synthase (NOS) inhibitor (nitro-L-arginine methyl ester, L-NAME) treatment on blood pressure (BP), heart rate (HR) and cardiac oxidant/antioxidant systems in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control (SC), (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, s.c. for 8 weeks) and (4) ET+L-NAME. BP and HR were monitored with tail-cuff method. The animals were sacrificed 24 h after last treatments and hearts were isolated and analyzed. Physical conditioning significantly increased respiratory exchange ratio (RER), cardiac nitric oxide (NO) levels, NOS activity and endothelial (eNOS) and inducible (iNOS) protein expression. Training significantly enhanced cardiac glutathione (GSH) levels, GSH/GSSG ratio and up-regulation of cardiac copper/zinc-superoxide dismutase (CuZn-SOD), manganese (Mn)-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) activity and protein expression. Training also caused depletion of cardiac malondialdehyde (MDA) and protein carbonyls. Chronic L-NAME administration resulted in depletion of cardiac NO level, NOS activity, eNOS, nNOS and iNOS protein expression, GSH/GSSG ratio and down-regulation of cardiac CuZn-SOD, Mn-SOD, CAT, GSH-PX, glutathione-S-transferase (GST) activity and protein expression. Chronic L-NAME administration enhanced cardiac xanthine oxidase (XO) activity, MDA levels and protein carbonyls. These biochemical changes were accompanied by increases in BP and HR after L-NAME administration. Interaction of training and NOS inhibitor treatment resulted in normalization of BP, HR and up-regulation of cardiac antioxidant defense system. The data suggest that physical conditioning attenuated the oxidative injury caused by chronic NOS inhibition by up-regulating the cardiac antioxidant defense system and lowering the BP and HR in rats.