Chaperone-like activity and hydrophobicity of alpha-crystallin

alpha-Crystallin, a prominent member of small heat shock protein (sHsp) family and a major structural protein of the eye lens is a large polydisperse oligomer of two isoforms, alphaA- and alphaB-crystallins. Numerous studies have demonstrated that alpha-crystallin functions like a molecular chaperone in preventing the aggregation of various proteins under a wide range of stress conditions. The molecular chaperone function of alpha-crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. alpha-Crystallin selectively interacts with non-native proteins thereby preventing them from aggregation and helps maintain them in a folding competent state. It has been proposed and generally accepted that alpha-crystallin suppresses the aggregation of other proteins through the interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded substrate protein. However, a quantifiable relationship between hydrophobicity and chaperone-like activity remains a matter to be concerned about. On an attentive review of studies on alpha-crystallin chaperone-like activity, particularly the studies that have direct or indirect implications to hydrophobicity and chaperone-like activity, we found several instances wherein the correlation between hydrophobicity and its chaperone-like activity is paradoxical. We thus attempted to provide an overview on the role of hydrophobicity in chaperone-like activity of alpha-crystallin, the kind of evaluation done for the first time.     

Osmotic flow through asymmetric membrane: A means for controlled delivery of drugs with varying solubility

A nondisintegrating, controlled release, asymmetric membrane capsular system of flurbiprofen was developed and evaluated for controlled release of the drug to overcome some of its side effects. Asymmetric membrane capsules were prepared using fabricated glass mold pins by phase inversion process. The effect of different formulation variables was studied based on 2³ factorial design; namely, level of osmogen, membrane thickness, and level of pore former. Effects of polymer diffusibility and varying osmotic pressure on drug release were also studied. Membrane characterization by scanning electron microscopy showed an outer dense region with less pores and an inner porous region for the prepared asymmetric membrane. Differential scanning calorimetry studies showed no incompatibility between the drug and the excipients used in the study. In vitro release studies for all the prepared formulations were done (n=6). Statistical test (Dunnett multiple comparison test) was applied for in vitro drug release atP>.05. The best formulation closely corresponded to the extra design checkpoint formulation by a similarity (f2) value of 92.94. The drug release was independent of pH but dependent on the osmotic pressure of the dissolution medium. The release kinetics followed the Higuchi model and the mechanism of release was Fickian diffusion. Published: July 7, 2006     

Involvement of endothelin in morphine tolerance in neuroblastoma (SH-SY5Y) cells

Long-term use of morphine in pain management leads to adverse effects, such as development of antinociceptive tolerance. We have previously shown the involvement of central endothelin (ET) mechanisms in morphine analgesia and development of tolerance in vivo. The present study was conducted to investigate the in vitro mechanism of interaction of the ET(A) receptor antagonist, BMS182874, and morphine during acute and chronic morphine tolerance in SH-SY5Y cells. SH-SY5Y cells were exposed to acute and chronic treatment with vehicle, morphine, ET-1, BMS182874, or morphine plus BMS182874. Activation of G-protein-coupled receptors in SH-SY5Y cells was determined using [35S]GTPgammaS binding assays. Acute morphine treatment produced a concentration-dependent increase in GTP binding. Median effective concentration (EC50) values were significantly decreased after acute morphine treament, suggesting sensitization of opioid receptors. Chronic morphine treatment produced a lower maximal response of GTP binding compared with both control (vehicle treated) and acute morphine treatment, indicating uncoupling of G-proteins. Acute and chronic exposure of cells to ET-1 did not affect changes in ET-1-induced GTP binding. BMS182874 treatment alone (acute or chronic) did not produce G-protein activation. However, in cells chronically cotreated with 10 microM morphine and 1 microM BMS182874, morphine-induced GTP stimulation was significantly higher than control (vehicle treated). The EC50 value after control treatment was 414 nM, and was significantly increased in chronically morphine-treated cells (>1000 nM ). However, the EC50 value in cells receiving a chronic treatment of BMS182874 and 63 nM morphine was significantly reduced compared with control (vehicle treated) and chronic morphine treatment. ET(A) antagonists significantly enhance the coupling of G-protein to opioid receptors. Therefore, we propose that restoration of morphine antinociception by ET(A) antagonists in morphine-tolerant animals is likely via a G-protein mediated mechanism.     

Proteomic analysis of mice hippocampus in simulated microgravity environment

Space travel induces many deleterious effects on the flight crew due to the '0' g environment. The brain experiences a tremendous fluid shift, which is responsible for many of the detrimental changes in physical behavior seen in astronauts. It therefore indicates that the brain may undergo major changes in its protein levels in a '0' g environment to counteract the stress. Analysis of these global changes in proteins may explain to better understand the functioning of brain in a '0' g condition. Toward such an effort, we have screened proteins in the hippocampus of mice kept in simulated microgravity environment for 7 days and have observed a few changes in major proteins as compared to control mice. Essentially, the results show a major loss of proteins in the hippocampus of mice subjected to simulated microgravity. These changes occur in structural proteins such as tubulin, coupled with the loss of proteins involved in metabolism. This preliminary investigation leads to an understanding of the alteration of proteins in the hippocampus in response to the microgravity environment.     

A new class of Gd-based DO3A-ethylamine-derived targeted contrast agents for MR and optical imaging

Synthetic bifunctional probes based on [4,7-bis-carboxymethyl-10-(2-aminoethyl)-1,4,7,10-tetraaza-cyclododec-1-yl]-acetic acid (DO3A-ethylamine) preloaded with gadolinium were prepared for applications in targeted magnetic resonance imaging (MRI) and optical imaging. A convenient route of synthesis is reported, which allowed conjugation of this probe with biomolecules for the preparation of model MR contrast agents for targeted imaging. The conjugated probes have the following interesting properties: GdDO3A-ethylamido-biotin (Gd-9) can be used for targeted imaging using an avidin-biotin system. The fluorescent probe GdDO3A-ethylthiourea-fluorescein (Gd-12) is a bimodal compound, which can be used for both MR and optical imaging. The precursors, DO3A-ethylamidopropyl-maleimide and DO3A-ethyl-isothiocyanate contain a highly reactive moiety, which can interact with free SH-terminals and N-terminals of biological molecules, respectively. In vitro MR relaxivity studies were performed at 300 MHz using different concentrations and chemical environments. MR relaxivity for ligand Gd-9 at pH 7.4, r1 was (3.32 +/- 0.03) s(-1) mM(-1) and r2 was (5.02 +/- 0.14) s(-1) mM(-1). For the mixture of Gd-9 with avidin, at pH 7.4, relaxivity increased linearly with the avidin concentration. A relaxivity enhancement of 45% for r1 and more than 400% for r2 with respect to the unbound biotinylated Gd3+ complex was found at a ratio of 4:1. MR relaxivity for ligand Gd-12, r1 was (5.36 +/- 0.05) s(-1) mM(-1) at pH 7.4. Fluorescence microscopy and spectroscopy of Gd-12-labeled 3T3 mouse fibroblasts showed a concentration-dependent intracellular uptake, accompanied by a slight dose-dependent increase in toxicity up to 150 microM. MR studies on labeled cells indicated a contrast enhancement in both T1- and T2-weighted images by the internalized compound, with the effect being more pronounced in T2-weighted images. Our results indicate that DO3A-ethylamine is a multipurpose precursor, from which various targeted contrast agents can be synthesized after a single-step conjugation with organic/bioorganic molecules.     

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.     

In vitro degradation of wheat straw by anaerobic fungi from small ruminants

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.     

Synthesis and antifungal activity of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles

Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger (ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a-d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 microg/disc against A. fumigatus and 11.71 microg/disc against A. flavus and A. niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 microg/ml in case of A. fumigatus and 31.25 microg/ml with A. flavus and A. niger. The MIC90 value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml against A. fumigatus. The MIC90 values of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 microg/ml. The in vitro toxicity of the most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 microg/ml. The standard drug amphotericin B exhibited 100% lysis at a concentration of 37.5 microg/ml.     

Depeptidization efforts on P3-P2' alpha-ketoamide inhibitors of HCV NS3-4A serine protease: effect on HCV replicon activity

Depeptidization efforts of the P(3)-P(2) region of P(3) capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P(2) nitrogen and modification of the P(2)' carboxylic acid terminus were essential for activity in the replicon assay.     

Diarylacetylene piperidinyl amides as novel anxiolytics

N-Phenyl-2-[1-[3-(2-pyridinylethynyl)benzoyl]-4-piperidine]acetamide (9) and related piperidine acetamide derivatives have good oral activity in the elevated plus maze, an animal model predictive of clinical efficacy for the treatment of anxiety. Modest affinity was observed for the neurokinin NK-1 and 2 receptors, which are known to be involved in the regulation of mood and emotion.