Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site

gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.     

Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.