The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

Activation of RAW 264.7 cells by Astraeus hygrometricus-derived heteroglucan through MAP kinase pathway

Mushroom-derived polysaccharides like β-glucan are being investigated for therapeutic properties for a long time, but their mode of action of immunomodulatory properties is not well established. In the present study, a heteroglucan from Astraeus hygrometricus designated as AE2 is investigated for its macrophage stimulatory properties using RAW 264.7 cell line. An augmentation of nitric oxide production is observed in the presence of AE2 in a dose-dependent manner due to up-regulation of iNOS (inducible NO synthase) expression; hence NF κB (nuclear factor κB) pathway is investigated. RAW 264.7 cells endured phosphorylation of Ikk (IκB kinase) and subsequently NF κB is translocated to the nucleus. Further, the PKC (protein kinase C) level of the cells enhanced significantly. We also found that AE2 could induce the phosphorylation of p38 MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2), MEK (MAPK/ERK kinase) and JNK (c-Jun N-terminal kinase), whereas it failed to induce phosphorylation of JAK2 (Janus kinase 2) and STAT1. These results indicated that the macrophage activation by AE2 might be exerted, at least in part, via MAPKs (mitogen-activated protein kinases) pathway of signal transduction.     

Comparison of two ferns (Adiantum capillus-veneris Linn. and Microsorium punctatum (Linn.) Copel) for their Cr accumulation potential and antioxidant responses

Study was undertaken to compare Cr accumulation in two ferns (Adiantum capillus-veneris Linn. and Microsorium punctatum (Linn.) Copel) and the role of antioxidants were also investigated towards metal tolerance in order to assess the use of ferns in phytomediation/ phytostabilization. Different concentrations (0, 50, 100, 150 microg g(-1) dw) of Cr were added to fern planted in pot containing 1 kg soil. In both the ferns, Cr accumulation increased with increase in metal concentration and maximum accumulation of 800.5 microg g(-1) (fronds) and 1457.4 microg g(-1) (roots) in M. punctatum and 660.8 microg g(-1) (fronds) and 1259.6 microg g(-1) (roots) in A. capillus-veneris was recorded. The increase in the levels of malondialdehyde, antioxidants and antioxidant enzymes (superoxide dismutase, glutathione peroxidase) in A. capillus-veneris was less pronounced than M. punctatum under Cr exposure as compared to their respective controls. In view of less decrease in chlorophyll content and antioxidants along with higher accumulation of Cr in the fronds M. punctatum, is indicative of its higher tolerance towards Cr. However, bioaccumulation factor (concentration of Cr in fronds/concentration of Cr in the soil) of both the ferns was recorded > 1 which qualifies the plants as potential Cr hyperaccumulator and suitable for phytoremediaton.     

In vitro immunostimulatory properties of Abrus lectins derived peptides in tumor bearing mice

In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-γ and TNF-α indicating a Th1 type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers (CD25⁺ and CD71⁺). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice.     

Mycobacterium avium 104 deleted of the methyltransferase D gene by allelic replacement lacks serotype-specific glycopeptidolipids and shows attenuated virulence in mice

Mycobacterium avium is a major opportunistic pathogen of AIDS patients in the United States. The understanding of M. avium pathogenesis has been hampered by the inability to create gene knockouts by homologous recombination, an important mechanism for defining and characterizing virulence factors. In this study a functional methyltransferase D (mtfD) gene was deleted by allelic replacement in the M. avium strain 104. Methyltransferase D is involved in the methylation of glycopeptidolipids (GPLs); highly antigenic glycolipids found in copious amounts on the M. avium cell surface. Interestingly, the loss of mtfD resulted in M. avium 104 containing only the non-serotype specific GPL. Results also suggest that the mtfD encodes for a 3-O-methyltransferase. The absence of significant amounts of any serotype-specific GPLs as a consequence of mtfD deletion indicates that the synthesis of the core 3,4-di-O-methyl rhamnose is a prerequisite for synthesis of the serotype-specific GPLs. Macrophages infected with the mtfD mutant show elevated production of tumour necrosis factor-alpha (TNF-alpha) and RANTES compared to control infections. In addition, the M. avium 104 mtfD mutant exhibits decreased ability to survive/proliferate in mouse liver and lung compared to wild-type 104, as assessed by bacterial counts. Importantly, the mtfD mutant complemented with a wild-type mtfD gene maintained an infection level similar to wild-type. These experiments demonstrate that the loss of mtfD results in a M. avium 104 strain, which preferentially activates macrophages in vitro and shows attenuated virulence in mice. Together our data support a role for GPLs in M. avium pathogenesis.     

Pathways in the degradation of hydrolyzed alcohols of butyl benzyl phthalate in metabolically diverse Gordonia sp. strain MTCC 4818

In the present study, the metabolic pathways involved in the degradation of benzyl alcohol and 1-butanol, the hydrolyzed products of butyl benzyl phthalate, were investigated by the Gordonia sp. strain MTCC 4818. The strain can utilize both benzyl alcohol and 1-butanol individually as sole carbon sources, where benzyl alcohol was found to be metabolized via benzaldehyde, benzoic acid and catechol, which was further degraded by ortho-cleavage dioxygenase to cis,cis-muconic acid and subsequently to muconolactone leading to tricarboxylic acid cycle. On the other hand, 1-butanol was metabolized via butyraldehyde and butyric acid, which was channeled into the tricarboxylic acid cycle via the beta-oxidation pathway. Numbers of dehydrogenases, both NAD+-dependent and NAD+-independent, were found to be involved in the degradation of benzyl alcohol and 1-butanol, where several dehydrogenases exhibited relaxed substrate specificity. Both 2,3- and 3,4-dihydroxybenzoic acids were utilized by the test organism for growth and metabolized by the ortho-cleavage pathway by the cell-free extract of benzoate-grown cells, similar to catechol, suggesting possible broad substrate specificity of the ring cleavage dioxygenase. Moreover, the test organism can utilize various primary and secondary alcohols, aliphatic aldehydes and acids in the C2-C5 range besides n-hexadecane, 1,4-butanediol and cyclohexanol individually as the sole carbon sources indicating metabolic diversity in the Gordonia sp. strain MTCC 4818.