Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.     

Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 microM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12-log10 protections, respectively, at the dose of 50 mg/kg body weight.     

A compilation of Bioactive Compounds from Ayurveda

This review deals with the key bioactive compounds and the role of medicinal plants in Ayurvedic systems of medicine in India and their earlier investigation. There has been an increase in demand for the Phytopharmaceutical products of Ayurvĕda in Western countries, because of the fact that the allopathic drugs have more side effects. Many pharmaceutical companies are now concentrating on manufacturing of Ayurvĕdic Phytopharmaceutical products. Ayurvĕda is the Indian traditional system of medicine, which also deals about pharmaceutical science. Different type of plant parts used for the Ayurvedic formulation; overall out line of those herbal scenario and its future prospects for the scientific evaluation of medicinal plants used by traditional healers are also discussed. In India most of them, where Ayurvedic treatment is frequently used, for their ailments and provides instructions to local people how to prepare medicine from the herbs. As much as possible importance is also given for the taxonomic literature.     

Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine

Chemopreventive effect of S-allylcysteine (constituent of garlic) on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis was evaluated in Wistar rats. Significantly decreased lipid peroxidation products (thiobarbituric acid reactive substances-TBARS and lipid hydroperoxides) with increased level of reduced glutathione, increased activities of glutathione S-transferase, and glutathione peroxidase were observed in liver of NDEA-treated rats when compared with control rats. The activities of superoxide dismutase and catalase were significantly decreased in tumor tissue when compared with control. Administration of S-allylcysteine (SAC) showed the inhibition of tumor incidence, modulated the lipid peroxidation, and increased the reduced glutathione, glutathione-dependent enzymes, superoxide dismutase, and catalase in NDEA-induced carcinogenesis. From our results, we speculate that S-allylcysteine mediates its chemopreventive effects by modulating lipid peroxidation, GST stimulation, and by increasing the antioxidants. Hence SAC prevents cells from loss of oxidative capacity in NDEA-induced hepatocarcinogenesis.     

Purification, characterization and bactericidal activities of basic phospholipase A2 from the venom of Agkistrodon halys (Chinese pallas)

Agkistrodon snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this study, we used reverse-phase HPLC to purify PLA(2) from the venom of Agkistrodon halys. The enzyme named as AgkTx-II, a basic Asp49 PLA(2), has a molecular masses of 13,869.05. The amino acid sequence and molecular mass of AgkTx-II was identical to those of an Asp49 basic myotoxic PLA(2) previously isolated from this venom. Antibacterial activities were tested by susceptibility and broth-dilution assays. AgkTx-II exerted a potent antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. The MIC values of AgkTx-II ranged between 85 and 2.76muM and was most effective against S. aureus, P. vulgaris, P. mirabilis (MIC of 21.25muM) and B. pseudomallei (MIC of 10.25muM). This AgkTx-II rapidly killed S. aureus, P. vulgaris and B. pseudomallei in a dose-dependent manner. The effect of the AgkTx-II on bacterial membranes was evaluated by scanning and transmission electron microscopy. AgkTx-II caused morphological alterations apparent on their cellular surfaces, suggesting a killing mechanism based on membrane permeabilization and damage. Cytotoxicity was measured by XTT tetrazolium (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and lactate dehydrogenase (LDH) assays using U-937 cells (monocytes). The AgkTx-II did not affect cell viability up to 500muM concentrations but cell death was evident at 1000muM concentration after 24 and 48h. Furthermore, the repeated exposure of AgkTx-II (2-14muM) treated mice showed different tissue alterations, mainly at the brain and kidney; the toxicological potential of AgkTx-II remains to be elucidated. The AgkTx-II exhibits no hemolytic action even at high doses (10-100muM) in human erythrocytes. However, the AgkTx-II is believed to exert its bactericidal effect by permeabilizing the bacterial membrane by forming pores. In addition, the basic PLA(2) AgkTx-II displays a bactericidal effect, which may be either dependent or independent of catalysis.     

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

The potential impact of recent insights into proteomic changes associated with glaucoma

Introduction: Glaucoma, a major ocular neuropathy, is still far from being understood on a molecular scale. Proteomic workflows revealed glaucoma associated alterations in different eye components. By using state-of-the-art mass spectrometric (MS) based discovery approaches large proteome datasets providing important information about glaucoma related proteins and pathways could be generated. Corresponding proteomic information could be retrieved from various ocular sample species derived from glaucoma experimental models or from original human material (e.g. optic nerve head or aqueous humor). However, particular eye tissues with the potential for understanding the disease’s molecular pathomechanism remains underrepresented.

Areas covered: The present review provides an overview of the analysis depth achieved for the glaucomatous eye proteome. With respect to different eye regions and biofluids, proteomics related literature was found using PubMed, Scholar and UniProtKB. Thereby, the review explores the potential of clinical proteomics for glaucoma research.

Expert commentary: Proteomics will provide important contributions to understanding the molecular processes associated with glaucoma. Sensitive discovery and targeted MS approaches will assist understanding of the molecular interplay of different eye components and biofluids in glaucoma. Proteomic results will drive the comprehension of glaucoma, allowing a more stringent disease hypothesis within the coming years.     

Characterization of human reflex tear proteome reveals high expression of lacrimal proline‐rich protein 4 (PRR4)

In‐depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC‐ESI‐MS/MS strategy for label‐free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC‐ESI‐MS/MS and targeted‐MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline‐rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland‐specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha‐1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.