Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial for their long-term function. The interaction between cells and extra-cellular components is indispensable in determining cellular behavior in tissues and on biomaterials. The fibrin that contains fibronectin shows promise in most aspects as a tissue engineering scaffold, whereas, deposition of elastin and collagen by endothelial cells grown in the lumen of the construct is desirable to improve post implant retention, mechanical stability and vaso-responsiveness. So far there is no report on production of extra-cellular matrix (ECM) proteins, elastin and collagen by endothelial cells (EC) in in vitro culture conditions. In this study, we have used a biomimetic approach of providing multiple growth factors (GF) in the fibronectin (FN)-containing fibrin matrix to induce production of elastin and collagen by the endothelial cells for application in vascular tissue engineering. Deposition of elastin and collagens with matrix remodeling is demonstrated through qualitative analysis of the matrices that were recovered after growing cells on the initial fibrin-FN-GF matrix. Expressions of mRNA for both proteins were assessed by real time polymerase chain reaction (RT-PCR) to estimate the effects of multiple growth factor compositions. Marked deposition of elastin and collagen was evidenced by staining the recovered matrix after different culture intervals. Obviously, the biomimetic environment created by adding angiogenic and platelet growth factors in the fibrin-fibronectin-gelatin matrix can induce deposition of collagens and elastin by EC.     

Feasibility of Provider-Initiated HIV Testing and Counselling of Tuberculosis Patients Under the TB Control Programme in Two Districts of South India

Background

Provider-initiated HIV testing and counselling (PITC) is internationally recommended for tuberculosis (TB) patients, but the feasibility, effectiveness, and impact of this policy on the TB programme in India are unknown. We evaluated PITC of TB patients across two districts in India considered to have generalized HIV epidemics, Tiruchirappalli (population 2.5 million) and Mysore (population 2.8 million).

Methodology/Principal Findings

Starting June 2007, healthcare providers in both districts were instructed to ascertain HIV status for all TB patients, and refer those with unknown HIV status to the nearest Integrated Counselling and Testing Centre (ICTC)—often in the same facility—for counselling and voluntary HIV testing. All TB patients registered from June 2007 to March 2008 were followed prospectively. Field investigators assessed PITC practices and abstracted data from routine TB programme records and HIV counselling registers to determine the proportion of TB patients appropriately evaluated for HIV infection. Patient records were traced to determine the efficiency of referral links to HIV care and antiretroviral treatment (ART). Between July 2007 and March 2008, 5299 TB patients were registered in both study districts. Of the 4701 with unknown HIV status at the time of TB treatment initiation, 3368 (72%) were referred to an ICTC, and 3111 (66%) were newly tested for HIV. PITC implementation resulted in the ascertainment of HIV status for 3709/5299 (70%) of TB patients, and detected 200 cases with previously undiagnosed HIV infection. Overall, 468 (8.8%) of all registered TB patients were HIV-infected; 177 (37%) were documented to have also received any ART.

Conclusions

With implementation of PITC in India, HIV status was successfully ascertained for 70% of TB patients. Previously undiagnosed HIV-infection was detected in 6.4% of those TB patients newly tested, enabling referral for life-saving anti-retroviral treatment. ART uptake, however, was poor, suggesting that PITC implementation should include measures to strengthen and support ART referral, evaluation, and initiation.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.     

Purification and assay protocols for obtaining highly active Jumonji C demethylases

Jumonji C (JmjC) lysine demethylases (KDMs) are Fe(II)-dependent hydroxylases that catalyze the oxidative demethylation of methyllysine residues in histones and nonhistone proteins. These enzymes play vital roles in regulating cellular processes such as gene expression, cell cycle progression, and stem cell self-renewal and differentiation. Despite their biological importance, recombinant forms of JmjC KDMs generally display low enzymatic activity and have remained challenging to isolate in a highly active form. Here we present a simple affinity purification scheme for Strep(II)-tagged JmjC KDMs that minimizes contamination by transition state metal ions, yielding highly active and pure enzyme. We also describe an optimized continuous fluorescent assay for KDMs that detects formaldehyde production during demethylation via a coupled reaction using formaldehyde dehydrogenase. Purification and kinetic analysis of the human KDMs JMJD2A and JMJD2D using these methods yielded activities substantially higher than those previously reported for these enzymes, which are comparable to that of the flavin-dependent KDM LSD1. In addition, we show that JMJD2A exhibited a lower catalytic efficiency toward a histone peptide bearing a chemically installed trimethyllysine analog compared with a bona fide trimethylated substrate. The methodology described here is broadly applicable to other JmjC KDMs, facilitating their biochemical characterization and high-throughput screening applications.     

Expression and localization of CLC chloride transport proteins in the avian retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.     

Cytological evidence on the origin of sweet potato

Summary The results of intensive meiotic studies, particularly of the karyology and chromosomal homology at the pachytene stage, in the sweet potato (Ipomoea batatas L.), which is a hexaploid (2 n = 90), have thrown considerable light on its origin and genome relationships. Using suitable criteria, such as relative length of chromosomes, centromere position, chromomere pattern, absence of light staining segments in one of the arms, presence of telochromomere etc., 40 of the 45 haploid chromosome complement at pachytene were identified and assigned to 19 chromosomal types. Among these types, eight were present singly; in six of the types, chromosomes were present in duplicate, and in two types, in triplicate. The occurrence of higher multivalent chromosomal associations such as hexavalents and pentavalents, in addition to the quadrivalents already reported, was recorded for the first time at the pachytene and metaphase I stages. The hexavalents at pachytene were resolved into three distinct types based on the morphology of the participating chromosomes. A maximum number of nine quadrivalents at the metaphase I stage and four in the incompletely analyzed pachytene nuclei were recorded. The constituent chromosomes of three of the quadrivalents at pachytene stage were identified. From these observations, it is suggested that (i) the three parental genomes are partly homologous (ii) two of the genomes show closer homology to one another than to the third and (iii) the three genomes differ with respect to one or more of the eight chromosomal types occurring singly. The available information rules out an autopolyploid origin for sweet potato and suggests that the parental genomes are from closely related taxa. The advantages are emphasized of pursuing similar studies in other American Ipomoea species to unravel their relationship with the sweet potato. Among other meiotic irregularities, a translocated chromosome and a chromosome carrying inversion were detected at the pachytene stage and the possible role they may play in varietal differentiation is discussed.     

Design,parallel synthesis,and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC₅₀ value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship.     

Multistep Aggregation Pathway of Human Interleukin-1 Receptor Antagonist: Kinetic, Structural, and Morphological Characterization

The complex, multistep aggregation kinetic and structural behavior of human recombinant interleukin-1 receptor antagonist (IL-1ra) was revealed and characterized by spectral probes and techniques. At a certain range of protein concentration (12-27 mg/mL) and temperature (44-48°C), two sequential aggregation kinetic transitions emerge, where the second transition is preceded by a lag phase and is associated with the main portion of the aggregated protein. Each kinetic transition is linked to a different type of aggregate population, referred to as type I and type II. The aggregate populations, isolated at a series of time points and analyzed by Fourier-transform infrared spectroscopy, show consecutive protein structural changes, from intramolecular (type I) to intermolecular (type II) β-sheet formation. The early type I protein spectral change resembles that seen for IL-1ra in the crystalline state. Moreover, Fourier-transform infrared data demonstrate that type I protein assembly alone can undergo a structural rearrangement and, consequently, convert to the type II aggregate. The aggregated protein structural changes are accompanied by the aggregate morphological changes, leading to a well-defined population of interacting spheres, as detected by scanning electron microscopy. A nucleation-driven IL-1ra aggregation pathway is proposed, and assumes two major activation energy barriers, where the second barrier is associated with the type I → type II aggregate structural rearrangement that, in turn, serves as a pseudonucleus triggering the second kinetic event.