Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O(2).

Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O(2)] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O(2) with moderate affinity, 135 and 222 µM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O(2)-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.

Mesalazine granules 1.5 g once-daily maintain remission in patients with ulcerative colitis who switch from other 5-ASA formulations: a pooled analysis from two randomised controlled trials.

BACKGROUND: Mesalazine (mesalamine) granules (MG) were shown to be effective for the maintenance of remission of ulcerative colitis (UC) in two double-blind placebo-controlled trials. AIM: To evaluate the efficacy of once-daily MG for maintenance of remission in patients with UC who switched from other 5-aminosalicylic acid (5-ASA) formulations. METHODS: Data from two independent multicenter, randomised, double-blind, placebo-controlled, 6-month trials evaluating patients with UC in remission were combined for analysis of a subpopulation of patients who switched from other 5-ASA formulations to MG 1.5 g or placebo upon randomisation. The primary endpoint was the percentage of patients who remained relapse-free at Month 6 or end of treatment. Relapse was defined as a Sutherland Disease Activity Index (SDAI) rectal bleeding score ≥1 and mucosal appearance score ≥2, a UC flare or medication used to treat a UC flare. RESULTS: Of the 487 patients who received 5-ASA maintenance therapy at enrolment, 322 were in the MG group and 165 were in the placebo group. The percentage of patients who remained relapse-free (based on Sutherland Disease Activity Index scores) after 6 months was significantly higher with MG than placebo (78.3% vs. 58.8%, P < 0.001). Rectal bleeding, stool frequency and the physician's rating of disease activity remained unchanged after 6 months in a higher percentage of patients using MG compared with those on placebo (P < 0.004 for each endpoint). CONCLUSION: Mesalazine granules 1.5 g once-daily is effective for maintenance of remission in UC patients who switch from other 5-ASA formulations. ClinicalTrials.gov identifiers NCT00744016, NCT00767728.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Stereoselective first total synthesis, confirmation of the absolute configuration and bioevaluation of botryolide-E.

A simple, first stereoselective total synthesis of botryolide-E has been described. The synthesis started from propylene oxide employing Jacobsen's hydrolytic kinetic resolution (HKR), selective epoxide opening, sharpless asymmetric dihydroxylation, one pot acetonide deprotection and lactonization as key steps. Further, the synthesis confirms the absolute configuration of the natural product botryolide-E and we evaluated the biological behavior of natural product botryolide-E against a panel of bacteria and fungi. Botryolide-E exhibits significant potent activity against Staphylococcus aureus (MTCC 96) (6.25 µg/ml), good against Escherichia coli (MTCC 443) (12.5 µg/ml), Bacillus subtilis (MTCC 441) (25 µg/ml) and compound 1 exhibited good to moderate antifungal activity.

Clinical trial: once-daily mesalamine granules for maintenance of remission of ulcerative colitis - a 6-month placebo-controlled trial.

BACKGROUND: Ulcerative colitis (UC) is a chronic relapsing and remitting idiopathic inflammatory bowel disorder. AIM: To evaluate once-daily mesalamine (mesalazine) granules (MG) for maintenance of remission of UC. METHODS: Randomized, double-blind, placebo-controlled trial of patients (n=209 MG, n=96 placebo) with UC in remission [revised Sutherland Disease Activity Index (SDAI) rectal bleeding=0, mucosal appearance <2] who took MG 1.5 g or placebo once-daily for up to 6 months. Primary efficacy endpoint: the percentage of patients who remained relapse-free at month 6/end of treatment. Relapse was defined as SDAI rectal bleeding score ≥1 and a mucosal appearance score ≥2, a UC flare, or initiation of medication to treat a UC flare. RESULTS: The percentage of relapse-free patients at month 6/end of treatment was higher with MG than placebo (78.9% vs. 58.3%, P < 0.001) in the intent-to-treat analysis. Significant differences (P ≤ 0.025) favouring MG were observed for most secondary endpoints including improvement in rectal bleeding, physician's disease activity rating, stool frequency, the SDAI at month 6/end of treatment, patients classified as a treatment success and relapse-free duration. The incidence of adverse events was similar between groups. CONCLUSIONS: Once-daily mesalamine (mesalazine) was effective in maintaining remission of UC for 6 months.

Stereoselective synthesis and biological evaluation of (R)-rugulactone, (6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one and its 4S epimer.

A simple and highly efficient synthetic route has been developed for synthesis of (R)-rugulactone (1a), (6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one (1b) and its 4S epimer 1c by employing proline-catalyzed alpha-aminooxylation, Sharpless epoxidation, Mitsunobu reaction as chirality introuducing steps. The antibacterial and antifungal activity of the compounds 1a, 1b and 1c were evaluated. 1a and 1b showed better antibacterial activity against Pseudomonas aeroginosa (MIC=12.5 microg/ml for 1a, 25 microg/ml for 1b) Klebsiella pneumonia (MIC=25 microg/ml for 1a). Compounds (1a, 1b, 1c) exhibited good to moderate antifungal activity.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA.

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation. We demonstrate that VINC interacts with the paraspeckle protein, P54nrb through three different protein interaction regions (PIRs) one of which (PIR-1) is localized near the 5' end while the other two (PIR-2, PIR-3) are localized near the 3' region of VINC. Our studies suggest that VINC may interact with P54nrb through a novel mechanism which is different from that reported for protein coding RNAs.

Click chemistry: studies on the synthesis of novel fluorous tagged triazol-4-yl substituted quinazoline derivatives and their biological evaluation--theoretical and experimental validation.

The formation of N- and O-propargylated quinazoline derivatives 2, 3 from quinazol-4-ones 1 was theoretically predicted by optimizations at B3LYP/6-31G* level, analysed kinetically and thermodynamically. Theoretical predictions are validated by experiment to observe the trends and found deviation. Thus, compound 1 was propargylated in basic media to obtain compound 2 and 3 in definite proportions. Each compound was further subjected to [3+2] cycloaddition using perfluoroalkyl azides through Click reaction under Sharpless conditions, and obtained a series of novel perfluoroalkyl-1H,1,2,3-triazol-4-yl substituted quinazolines 4, 5, and 6. All the compounds were screened for antimicrobial activity and identified potential compounds.

Synthesis, photochemical E (trans)-->Z (cis) isomerization and antimicrobial activity of 2-chloro-5-methylpyridine-3-olefin derivatives.

2-Chloro-5-methylpyridine-3-olefin derivatives (3a-e) have been synthesized from 2-chloro-5-methylnicotinaldehyde (1) and studied their photochemical E (trans)-->Z (cis) isomerization upon direct irradiation and triplet sensitized excitation for the first time. The triplet sensitized excitations of the compounds yielded high Z (4a-e) isomer composition, whereas the direct excitation results in less Z (4a-e) isomer composition, indicating triplet pathway is very efficient in converting the E (trans)-->Z (cis) isomer. Thus synthesized E (3a-c and 3e) and generated Z (4a-c and 4e) isomers were tested for antimicrobial activity. Antifungal activity of these pyridine derivatives are closely comparable to the standard used.

An efficient synthesis of bis(indolyl)methanes and evaluation of their antimicrobial activities.

A versatile and efficient method has been developed for the synthesis of bis(indolyl)methanes by using aluminium triflate (0.5 mol%) as a novel catalyst. Further, some of the synthesized compounds were evaluated for their efficacy as antibacterial and antifungal activities. Most of the compounds have shown moderate to good inhibitory activity.

Design, synthesis, structure-activity relationship and antibacterial activity series of novel imidazo fused quinolone carboxamides.

A series of novel 7,8 and 1,8 imidazo fused quinolone carboxamides are synthesized and evaluated against antibacterial activity. 1,8 Imidazo fused quinolones exhibit moderate antibacterial activity. Molecular modeling studies were carried out to optimize the pharmacophore.

GdCl(3) catalysed Grieco condensation: a facile approach for the synthesis of novel pyrimidine and annulated pyrimidine fused indazole derivatives in single pot under mild conditions and their anti-microbial activity.

Indazole regioisomers such as 3-amino-4-(trifluoromethyl)-6-phenyl-1H-indazole-7-carbonitrile 1 and 3-amino-6-(trifluoromethyl)-4-phenyl-1H-indazole-7-carbonitrile 2 were independently reacted with formaldehyde followed by unsymmetrical, symmetrical and cyclic electron rich olefins in presence of GdCl(3) as catalyst and obtained pyrimidine fused indazole derivatives 3 and 4, respectively. The reaction is found to be concerted and an exclusive product is formed. Representative examples of compounds 3 and 4 were screened against Gram-positive, Gram-negative bacteria and fungal species such as yeast and filamentous fungi in vitro. Compound 3f showed significant activity against all species of Gram-positive and Gram-negative bacteria, whereas compounds 3h and 4a showed the least activity with reference to penicillin as well as streptomycin. Similarly compound 3c showed promising activity against yeast and filamentous fungi whereas compound 3f is inactive at the maximum concentration of 150 microg/mL.

Anti-tubercular agents. Part IV: Synthesis and antimycobacterial evaluation of nitroheterocyclic-based 1,2,4-benzothiadiazines.

In continuation of our earlier work on benzothiadiazines, we have prepared a series of nitrofuran, nitrothiophene and arylfuran coupled benzothiadiazines and evaluated them for antimycobacterial and antibacterial activities. One of the compounds 2f has shown good in vitro antimycobacterial activity. All the synthesized compounds have shown moderate to good antibacterial activity.

Characterization of Arabidopsis thaliana SufE2 and SufE3: functions in chloroplast iron-sulfur cluster assembly and Nad synthesis.

In this study we characterize two novel chloroplast SufE-like proteins from Arabidopsis thaliana. Other SufE-like proteins, including the previously described A. thaliana CpSufE, participate in sulfur mobilization for Fe-S biosynthesis through activation of cysteine desulfurization by NifS-like proteins. In addition to CpSufE, the Arabidopsis genome encodes two other proteins with SufE domains, SufE2 and SufE3. SufE2 has plastid targeting information. Purified recombinant SufE2 could activate the cysteine desulfurase activity of CpNifS 40-fold. SufE2 expression was flower-specific and high in pollen; we therefore hypothesize that SufE2 has a specific function in pollen Fe-S cluster biosynthesis. SufE3, also a plastid targeted protein, was expressed at low levels in all major plant organs. The mature SufE3 contains two domains, one SufE-like and one with similarity to the bacterial quinolinate synthase, NadA. Indeed SufE3 displayed both SufE activity (stimulating CpNifS cysteine desulfurase activity 70-fold) and quinolinate synthase activity. The full-length protein was shown to carry a highly oxygen-sensitive (4Fe-4S) cluster at its NadA domain, which could be reconstituted by its own SufE domain in the presence of CpNifS, cysteine and ferrous iron. Knock-out of SufE3 in Arabidopsis is embryolethal. We conclude that SufE3 is the NadA enzyme of A. thaliana, involved in a critical step during NAD biosynthesis.

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

Synthesis and antibacterial activity of various substituted s-triazines.

Series of substituted-s-triazines (1-22) were synthesized and evaluated for their in vitro antibacterial activity against six representative Gram-positive and Gram-negative bacterial strains. Many compounds have displayed comparable antibacterial activity against Bacillus sphaericus and significantly active against other tested organisms with reference to streptomycin.

Convenient synthesis of novel 4-substitutedamino-5-trifluoromethyl-2,7-disubstituted pyrido[2,3-d] pyrimidines and their antibacterial activity.

Novel pyrido[2,3-d]pyrimidines 4 have been synthesised starting from 2-amino-4-trifluoromethyl-6-substituted nicotinonitriles 1 via imine formation, selective amination followed by Dimroth rearrangement. Compounds 4 were screened against Gram +ve and -ve bacteria in vitro. Compounds 4h and 4d showed significant activity against all species of Gram positive bacteria and moderate activity against Gram negative bacteria. N-2,4 difluorophenyl compounds 4l and 4m were the least active among all the compounds. All the compounds were inactive against Pseudomonas aeruginosa at the maximum concentration of 200 microg ml(-1).

Synthesis of multisubstituted quinolines from Baylis-Hillman adducts obtained from substituted 2-chloronicotinaldehydes and their antimicrobial activity.

Baylis-Hillman acetates were synthesized from substituted 2-chloronicotinaldehydes and were conveniently transformed into multisubstituted quinolines and cyclopenta[g]quinolines on reaction with nitroethane or ethyl cyanoacetate via a successive S(N)2'-S(N)Ar elimination strategy. Thus, synthesized quinolines were evaluated for antimicrobial activity and found having substantial antibacterial and antifungal activity.

Synthesis and biological evaluation of novel C (7) modified chrysin analogues as antibacterial agents.

The natural product, chrysin (5,7-dihydroxy flavone), obtained from Oroxylum indicum, exhibits numerous biological activities including anticancer, anti-inflammatory, and antiallergic activities. Three series of chrysin analogues were prepared, in which chrysin and heterocyclic moieties are separated by 3-carbon, 4-carbon, and 6-carbon spacers. All the derivatives were screened for antibacterial activity against a panel of susceptible and resistant Gram-positive and Gram-negative organisms. It was observed that most of the derivatives displayed significant activity as compared to their parent compound (chrysin).