Diaryloxy methano phenanthrenes: a new class of antituberculosis agents

A new series of diaryloxy methano phenanthrenes were prepared through tertiary-aminoalkylations of [(methoxy-phenyl)-phenanthren-9-yl-methyl]-phenols obtained from Friedel-Crafts alkylations on (methoxy-phenyl)-phenanthren-9-yl-methanols. These series of compounds were evaluated against Mycobacterium tuberculosis H37Rv and showed the desired activity in the range of 6.25 microg/mL in vitro. One of the compound 12j protects the mice from the challenge of M. tuberculosis in vivo, as 30% of the mice were survived at treatment of 50 mg/kg body weight.     

A novel potassium deficiency-induced stimulon in<Emphasis Type="Italic">Anabaena torulosa</Emphasis>

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacteriumAnabaena torulosa and of nine proteins inEscherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K⁺ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase ofA. torulosa and the glycine betaine-binding protein ofE. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria.     

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya

Soil samples collected from two hot springs, Soldhar and Ringigad, both located in the Garhwal region of Uttaranchal Himalaya were analysed for their physical, chemical and microbial components. The alkaline pH, total absence of carbon and nitrogen, and high temperature were features common to soil samples from both sites. The Soldhar samples contained higher amounts of Cu, Fe and Mn. Ringigad soil was devoid of Cu, but had much higher phosphate. While the optimum incubation temperature for isolating the maximum microbial counts from soil samples from the two sites was 50 degrees C, microbial growth in broth was also observed when incubated at 80 degrees C. Microscopic examination revealed three types of microbial populations, i.e., bacteria, yeast and filamentous organisms. The soil samples were found to be dominated by spore forming rods. Out of 58 aerobic isolates, 53 were gram positive bacilli. Gram positive anaerobic oval rods were also observed up to 60 degrees C. Soil dilution plates revealed the presence of antagonistic and phosphate solubilizing populations.     

Meiosis-specific yeast Hop1 protein promotes synapsis of double-stranded DNA helices via the formation of guanine quartets

In most eukaryotes, genetic exchange between paired homologs occurs in the context of a tripartite proteinaceous structure called the synaptonemal complex (SC). Genetic analyses have revealed that the genes encoding SC proteins are vital for meiotic chromosome pairing and recombination. However, the number, nature and/or the mechanism used by SC proteins to align chromosomes are yet to be clearly defined. Here, we show that Saccharomyces cerevisiae Hop1, a component of SC, was able to promote pairing of double-stranded DNA helices containing arrays of mismatched G/G sequences. Significantly, pairing was rapid and robust, independent of homology in the arms flanking the central G/G region, and required four contiguous guanine residues. Furthermore, data from truncated DNA double helices showed that 20 bp on either side of the 8 bp mismatched G/G region was essential for efficient synapsis. Methylation interference indicated that pairing between the two DNA double helices involves G quartets. These results suggest that Hop1 is likely to play a direct role in meiotic chromosome pairing and recombination by its ability to promote synapsis between double-stranded DNA helices containing arrays of G residues. To our knowledge, Hop1 is the first protein shown to promote synapsis of DNA double helices from yeast or any other organism.     

Modulation of renal disease in MRL/lpr mice by suberoylanilide hydroxamic acid

Epigenetic regulation of gene expression is involved in the development of many diseases. Histone acetylation is a posttranslational modification of the nucleosomal histone tails that is regulated by the balance of histone deacetylases and histone acetyltransferases. Alterations in the balance of histone acetylation have been shown to cause aberrant expression of genes that are a hallmark of many diseases, including systemic lupus erythematosus. In this study, we determined whether suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor: 1) inhibits inflammatory mediator production in vitro and 2) modulates lupus progression in vivo. Mesangial cells isolated from 10-wk-old MRL/lpr mice were stimulated with LPS/IFN-gamma and incubated with SAHA. TNF-alpha, IL-6, NO, and inducible NO synthase expression were inhibited by SAHA. We then treated MRL/lpr mice with daily injections of SAHA from age 10 to 20 wk. The animals treated with SAHA had decreased spleen size and a concomitant decrease in CD4-CD8- (double-negative) T cells compared with controls. Serum autoantibody levels and glomerular IgG and C3 deposition in SAHA-treated mice were similar to controls. In contrast, proteinuria and pathologic renal disease were significantly inhibited in the mice receiving SAHA. These data indicate that SAHA blocks mesangial cell inflammatory mediator production in vitro and disease progression in vivo in MRL/lpr mice.     

Double belt structure of discoidal high density lipoproteins: molecular basis for size heterogeneity

We recently proposed an all-atom model for apolipoprotein (apo) A-I in discoidal high-density lipoprotein in which two monomers form stacked antiparallel helical rings rotationally aligned by interhelical salt-bridges. The model can be derived a priori from the geometry of a planar bilayer disc that constrains the hydrophobic face of a continuous amphipathic alpha helix in lipid-associated apoA-I to a plane inside of an alpha-helical torus. This constrains each apoA-I monomer to a novel conformation, that of a slightly unwound, curved, planar amphipathic alpha 11/3 helix (three turns per 11 residues). Using non-denaturing gradient gel electrophoresis, we show that dimyristoylphosphocholine discs containing two apoA-I form five distinct particles with maximal Stokes diameters of 98 A (R2-1), 106 A (R2-2), 110 A (R2-3), 114 A (R2-4) and 120 A (R2-5). Further, we show that the Stokes diameters of R2-1 and R2-2 are independent of the N-terminal 43 residues (the flexible domain) of apoA-I, while the flexible domain is necessary and sufficient for the formation of the three larger complexes. On the basis of these results, the conformation of apoA-I on the R2-2 disc can be modeled accurately as an amphipathic helical double belt extending the full length of the lipid-associating domain with N and C-terminal ends in direct contact. The smallest of the discs, R2-1, models as the R2-2 conformation with an antiparallel 15-18 residue pairwise segment of helixes hinged off the disc edge. The conformations of full-length apoA-I on the flexible domain-dependent discs (R2-3, R2-4 and R2-5) model as the R2-2 conformation extended on the disc edge by one, two or three of the 11-residue tandem amphipathic helical repeats (termed G1, G2 and G3), respectively, contained within the flexible domain. Although we consider these results to favor the double belt model, the topographically very similar hairpin-belt model cannot be ruled out entirely.     

A role for sorting nexin 2 in epidermal growth factor receptor down-regulation: evidence for distinct functions of sorting nexin 1 and 2 in protein trafficking

Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps)5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.     

Effect of aqueous leaf extract of Azadirachta indica on the reproductive organs in male mice

Effect of oral administration (50, 100, and 200 mg/kg body weight/day, for 28 days) of aqucous leaf extract of neem (Azadirachta indica) on the male reproductive organs of the Parkes (P) strain mice was investigated. The treatment had no effect on body weight and the reproductive organs weight. In treated mice, testes showed both normal and affected seminiferous tubules in the same sections; the affected seminiferous tubules showed intraepithelial vacuolation, loosening of germinal epithelium, marginal condensation of chromatin in round spermatids, occurrence of giant cells, mixing of germ cell types in stages of spermatogenesis and degenerated appearance of germ cells. In severe cases, the tubules were lined with Sertoli cells only, Sertoli cells and rare germ cells, or with Sertoli cells and several germ cells but without cellular association patterns. Also, the frequency of affected seminiferous tubules in testes of the extract-treated mice was significantly higher than the controls, though this remained unaffected in mice treated at 50 mg/kg body weight of the extract. Doses at 50 or 100 mg/kg body weight of neem leaf extract did not cause appreciable alterations in histological appearance of the epididymis, while a dose of 200 mg/kg body weight caused marked alterations both in histological appearance and the level of sialic acid in the duct. The treatment also had adverse effects on motility, morphology, and number of spermatozoa in the cauda epididymidis, level of fructose in the seminal vesicle, and on litter size. After 42 days of withdrawal of the treatment, the alterations induced in the reproductive organs recovered to control levels. Our results suggested that treatment with neem leaf extract caused reversible alterations in the male reproductive organs of P mice.     

Ruthenium(II/III) mediated transformation of 1,2-bis(2′-pyridylmethyleneimino)benzene (L) to 2-(2′-benzimidazolyl)pyridine (L′H) and its in situ formed complexes with Ru(II): X-ray structure of trans-[Ru(PPh3)2(L′H)2](ClO4)2

A series of ruthenium (II) complexes of formulae trans-[Ru(PPh₃)₂(L′H)₂](ClO₄)₂ (1), [Ru(bpy)(L′H)₂](ClO₄)₂ (2), [Ru(bpy)₂(L′H)](ClO₄)₂ (3), cis-[Ru(DMSO)₂(L′H)₂]Cl₂ (4), and [Ru(L′H)₃](PF₆)₂ (5) (where L′H = 2-(2′-benzimidazolyl)pyridine) have been synthesized by reaction of the appropriate ruthenium precursor with 1,2-bis(2′-pyridylmethyleneimino)benzene (L). The complexes were characterized by elemental analyses, spectroscopic and electrochemical data. All the complexes were found to be diamagnetic and hence metal is in +2 oxidation state. The molecular structure of trans-[Ru(PPh₃)₂(L′H)₂](ClO₄)₂ has been determined by the single crystal X-ray diffraction studies. The molecular structure shows that Ru(II) is at the center of inversion of an octahedron with N₄P₂ coordination sphere. The ligand acts as a bidentate N,N′donor. The electronic spectra of the complexes display intense MLCT bands in the visible region.Cyclic voltammetric studies show quasi-reversible oxidative response at 0.99-1.32 V (vs Ag/AgCl reference electrode) due to Ru(III)/Ru(II) couple.     

Nitric oxide delays oocyte aging

Nitric oxide (NO) is a ubiquitous signaling molecule that plays a crucial role in oocyte maturation and embryo development. However, its role in oocyte aging is unclear. To examine how NO affects oocyte aging, we retrieved young and relatively old mouse oocytes and exposed them to increasing concentrations of NO donor S-nitroso acetyl penicillamine (SNAP). Aging related phenomena of ooplasmic microtubule dynamics (OMD), cortical granule (CG) exocytosis, zona pellucida (ZP) hardening, and spindle/chromatin integrity were studied at each SNAP concentration using fluorescence immunocytochemistry and confocal microscopy and compared with respective unexposed controls. Exposure of both young and old oocytes to NO resulted in a significant diminution in OMD and ZP dissolution time, whereas spontaneous CG loss decreased in old NO exposed oocytes compared to controls (P < 0.001 for all). Furthermore, NO exposure decreased the rate of spindle abnormalities in oocytes compared to unexposed controls. Interestingly, in old oocytes, the positive influence of NO was attenuated beyond 0.23 microM/min and disappeared at 0.46 microM/min NO. Overall, a significant dose-response relationship was noted between NO exposure and markers of aging with between 50 and 100 microM SNAP (0.11-0.23 microM/min NO, P < 0.0001). Collectively, our results demonstrate for the first time that exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus in young and old oocytes.