Enantioselective total synthesis of macrosphelides A and E

Enantioselective synthesis of 16-membered trilactone macrolides, macrosphelide A and E from (S)-lactic acid is described. Key features of the synthesis include the utility of a hitherto unexplored beta-ketophosphonate derived from lactic acid and Yamaguchi lactonization leading to the title compounds. (C) 2011 Elsevier Ltd. All rights reserved.

Total Synthesis of (-)-Anamarine

Total synthesis of polyhydroxy delta-pyranone natural product (-)-anamarine is accomplished from D-(-)-tartaric acid. The main feature of the synthesis is the utility of hitherto unexplored beta-keto phosphonate derived from tartaric acid amide and further elaboration involving stereoselective reduction.

Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Characterization of Major Zinc Containing Myonecrotic and Procoagulant Metalloprotease `Malabarin' from Non Lethal Trimeresurus malabaricus Snake Venom with Thrombin Like Activity: Its Neutralization by Chelating Agents

A major myonecrotic zinc containing metalloprotease `malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu-Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A alpha followed by B beta subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Photoactivated DNA cleavage and anticancer activity of pyrenyl-terpyridine lanthanide complexes

Lanthanide(III) complexes Ln(R-tpy)(acac)(NO3)(2)] (Ln = La(III) in 1, 2; Gd(III) in 4, 5) and Ln(py-tpy)(sacac)(NO3)(2)] (Ln = La(III), Gd(III), 6), where R-tpy is 4'-phenyl-2,2':6',2 `'-terpyridine (ph-tpy in 1, 4), 4'-(1-pyrenyl)-2,2':6',2 `'-terpyridine (py-tpy in 2, 3, 5 and 6), acac is acetylacetonate and sacac is 4-hydroxy-6-{4-(beta-D-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-on ate, were prepared to study their DNA photocleavage activity and photocytotoxicity. Complexes La(ph-tpy)(acac)(E-tOH)(NO3)(2)] (1a) and Gd(ph-tpy)(acac)(NO3)(2)] (4) were characterized by X-ray crystallography. The 1:1 electrolytic complexes bind to calf thymus DNA. The py-tpy complexes cleave pUC19 DNA and exhibit remarkable photocytotoxicity in HeLa cells in UV-A light of 365 nm with apoptotic cell death (IC50: similar to 40 nM in light, >200 mu M in dark). Confocal microscopy using HeLa cells reveal primarily cytosolic localization of the complexes. (C) 2012 Elsevier Masson SAS. All rights reserved.

The substrate dependent competitive formation of cyclobutanones and cyclopentanones in an intramolecular rhodium carbenoid CH insertion reaction: enantiospecific total synthesis of silphiperfol-6-ene

The enantiospecific total synthesis of silphiperfol-6-ene has been accomplished starting from the readily available monoterpene (R)-limonene, employing a rhodium carbenoid insertion into the CH bond of a tertiary methyl group. A substrate dependent competitive insertion of the rhodium carbenoid in the gamma- and beta-CH bonds to form cyclopentanone and cyclobutanones, respectively, has been described. (C) 2012 Elsevier Ltd. All rights reserved.

Caryophyllene-Rich Essential Oil of Didymocarpus tomentosa: Chemical Composition and Cytotoxic Activity

The essential oil from the leaves of Didymocarpus tomentosa was extracted by hydrodistillation and analyzed by GC/FID and GC/MS. Twenty five constituents amounting to 81.6% of the oil were identified. The leaf oil contained 78.7% sesquiterpenes and 2.9% monoterpenes. The leaf essential oil of D. tomentosa is a unique caryophyllene-rich natural source containing beta-caryophyllene, caryophyllene oxide, alpha-humulene and humulene oxide. The cytotoxic activity of the oil was determined by the BSLT using shrimp larva and the MTT assay using HeLa tumor cell line. The oil showed significant cytotoxic activity with LC50 and IC50 values of 12.26 and 11.4 mu g/mL, respectively. This is the first report on the chemical composition and cytotoxic activity of the essential oil of D. tomentosa.

Synthesis, morphology, optical and photocatalytic performance of nanostructured beta-Ga2O3

Fine powders of beta-Ga2O3 nanostructures were prepared via low temperature reflux condensation method by varying the pH value without using any surfactant. The pH value of reaction mixture had great influence on the morphology of final products. High crystalline single phase beta-Ga2O3 nanostructures were obtained by thermal treatment at 900 degrees C which was confirmed by X-ray diffraction and Raman spectroscopy. The morphological analysis revealed rod like nanostructures at lower and higher pH values of 6 and 10, while spindle like structures were obtained at pH = 8. The phase purity and presence of vibrational bands were identified using Fourier transform infrared spectroscopy. The optical absorbance spectrum showed intense absorption features in the UV spectral region. A broad blue emission peak centered at 441 nm due to donor-acceptor gallium-oxygen vacancy pair recombination appeared. The photocatalytic activity toward Rhodamine B under visible light irradiation was higher for nanorods at pH 10.

Anti-inflammatory activity of the ethanol extract of Dictamnus dasycarpus leaf in lipopolysaccharide-activated macrophages

Background: Dictamnus dasycarpus is widely used as a traditional remedy for the treatment of eczema, rheumatism, and other inflammatory diseases in Asia. The current study investigates the molecular mechanism of anti-inflammatory action of the ethanol extract of Dictamnus dasycarpus leaf (DE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methods: Nitric oxide (NO) production was assessed by Griess reaction and the mRNA and protein expressions of pro inflammatory cytokines, transcription factor, and enzymes were determined by real-time RT-PCR and immunoblotting analysis. Results: DE (0.5 and 1 mg/mL) suppressed the NO production by 10 and 33%, respectively, compared to the untreated group in LPS-stimulated RAW 264.7 cells. DE (0.5 and 1 mg/mL) reduced the mRNA expression of key transcription factor nuclear factor-kappa B by 7 and 24%, respectively compared to the untreated group in LPS activated macrophage. The pro inflammatory cytokines such as tumor necrosis factor a and interleukin 1 beta were also decreased by DE treatment. Moreover, the protein expression of pro inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase 2 were also dramatically attenuated by DE in a dose dependent manner. Conclusions: These results suggest that Dictamnus dasycarpus leaf has a potent anti-inflammatory activity and can be used for the development of new anti-inflammatory agents.

Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed `form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

Evolution of Aromatic beta-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

The bglA gene of Escherichia coli encodes phospho-beta-glucosidase A capable of hydrolyzing the plant-derived aromatic beta-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic beta-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bglA transcript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within the bglA structural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function

Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES-40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES-RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA-protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES-RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S-3 ` UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES-RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.

Optical properties and CCN activity of aerosols in a high-altitude Himalayan environment: Results from RAWEX-GVAX

The seasonality and mutual dependence of aerosol optical properties and cloud condensation nuclei (CCN) activity under varying meteorological conditions at the high-altitude Nainital site (2km) in the Indo-Gangetic Plains were examined using nearly year-round measurements (June 2011 to March 2012) at the Atmospheric Radiation Measurement mobile facility as part of the Regional Aerosol Warming Experiment-Ganges Valley Aerosol Experiment of the Indian Space Research Organization and the U.S. Department of Energy. The results from collocated measurements provided enhanced aerosol scattering and absorption coefficients, CCN concentrations, and total condensation nuclei concentrations during the dry autumn and winter months. The CCN concentration (at a supersaturation of 0.46) was higher during the periods of high aerosol absorption (single scattering albedo (SSA)<0.80) than during the periods of high aerosol scattering (SSA>0.85), indicating that the aerosol composition seasonally changes and influences the CCN activity. The monthly mean CCN activation ratio (at a supersaturation of 0.46) was highest (>0.7) in late autumn (November); this finding is attributed to the contribution of biomass-burning aerosols to CCN formation at high supersaturation conditions.

Imidazolium based ionic liquid type surfactant improves activity and thermal stability of lipase of Rhizopus oryzae

Lipase and surfactant together form a potent pair in various biotransformation, industrial application and biotechnological studies. The present investigation deals with changes in the activity, stability and structure of lipase from Rhizopus oryzae NRRL 3562 in presence of long chain ionic liquid-type imidazolium surfactant. Both the activity and stability were found to be enhanced in presence of the surfactant at low concentration (1-125 mu M) followed by inhibition at high concentration. The activity increased by 80% and thermal deactivation temperature raised by 2.5 degrees C. Investigations by ultraviolet-visible spectroscopy and circular dichroism revealed structural changes leading to rise in beta-sheet content and lowering of a-helix at low surfactant concentrations. Deactivation at high concentration correlated with greater structural changes depicted by spectroscopic studies. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature involving non-covalent interactions. High negative value of entropy signifies exposure of hydrophobic domains and increase in structural rigidity, which correlates with active site being more accessible and rigid in presence of the surfactant. Application of these surfactants hold greater potential in the field of lipase based biotransformations, enzyme structural modifications and studies. (C) 2015 Elsevier B.V. All rights reserved.

Structural insights into N-terminal to C-terminal interactions and implications for thermostability of a (beta/alpha)(8)-triosephosphate isomerase barrel enzyme

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)(8)-triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 degrees C to 75 degrees C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 degrees C to 68 degrees C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. Database The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively