Structural insights into the novel diadenosine 5',5‴-P¹,P⁴-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

Rv2613c is a diadenosine 5′,5?-P¹,P⁴-tetraphosphate (Ap₄A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (Ap_nA) hydrolases and Ap₄A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family Ap_nA hydrolases than to that of typical Ap₄A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with Ap_nA phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family Ap_nA hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap₄A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis.     

An Adaptor Molecule Afadin Regulates Lymphangiogenesis by Modulating RhoA Activity in the Developing Mouse Embryo

Afadin is an intracellular binding partner of nectins, cell-cell adhesion molecules, and plays important roles in the formation of cell-cell junctions. Afadin-knockout mice show early embryonic lethality, therefore little is known about the function of afadin during organ development. In this study, we generated mice lacking afadin expression in endothelial cells, and found that the majority of these mice were embryonically lethal as a result of severe subcutaneous edema. Defects in the lymphatic vessels of the skin were observed, although the morphology in the blood vessels was almost normal. Severe disruption of VE-cadherin-mediated cell-cell junctions occurred only in lymphatic endothelial cells, but not in blood endothelial cells. Knockout of afadin did not affect the differentiation and proliferation of lymphatic endothelial cells. Using in vitro assays with blood and lymphatic microvascular endothelial cells (BMVECs and LMVECs, respectively), knockdown of afadin caused elongated cell shapes and disruption of cell-cell junctions among LMVECs, but not BMVECs. In afadin-knockdown LMVECs, enhanced F-actin bundles at the cell periphery and reduced VE-cadherin immunostaining were found, and activation of RhoA was strongly increased compared with that in afadin-knockdown BMVECs. Conversely, inhibition of RhoA activation in afadin-knockdown LMVECs restored the cell morphology. These results indicate that afadin has different effects on blood and lymphatic endothelial cells by controlling the levels of RhoA activation, which may critically regulate the lymphangiogenesis of mouse embryos.     

Contribution of ferrous iron to maintenance of the gastric colonization of Helicobacter pylori in miniature pigs

Our previous study showed that the colonization levels of Helicobacter pylori were higher in the stomachs of 5-day-old miniature pigs than in 2-week-old ones. As dietary factors can cause these differences, we compared two diets, i.e., Weanymilk and a similar formula with a higher concentration of Fe(II), Weanylobulin. The colonization levels in the fundic mucosa were significantly higher in 2-week-old pigs fed Weanylobulin than in those fed Weanymilk. Supplementing Weanylobulin with an iron chelator, deferoxamine mesylate, significantly lowered the bacteria counts in the gastric mucosa. Normal diets supplemented with Fe(II) in 2-month-old pigs caused significantly more sites of bacteria in the antrum compared with normal diets alone. In addition, ranitidine, an inhibitor of gastric acid secretion that reduces Fe(III) to Fe(II) in the stomach, decreased the bacteria counts in 10-month-old pigs. These results suggested that Fe(II) maintained the colonization levels of H. pylori in the stomach of the miniature pigs.     

A Novel IgM‐capture enzyme‐linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection

An ELISA that measures anti‐PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG‐based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti‐PT IgG antibodies. To solve this problem, we developed a novel IgM‐capture ELISA that measures serum anti‐Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti‐Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti‐Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti‐Vag8 IgM‐capture ELISA. The results revealed that the anti‐Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti‐Vag8 IgM‐capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti‐PT IgG ELISA kit. Moreover, it was shown that anti‐Vag8 IgM antibodies were induced earlier than anti‐PT IgG antibodies on sequential patients’ sera. These data indicate that our novel anti‐Vag8 IgM‐capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

Interorganelle trafficking of ceramide is regulated by phosphorylation-dependent cooperativity between the PH and START domains of CERT

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositide-binding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.     

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.     

Pathological effect of synthetic cereulide, an emetic toxin of Bacillus cereus, is reversible in mice

Cereulide is the causative toxin of the emetic type of food-borne illness caused by Bacillus cereus. This toxin was previously shown to be associated with fulminant liver failure in a human case. Mice were injected i.p. with synthetic cereulide and the development of histopathological changes was examined. Hepatocytes showed mitochondrial swelling with loss of cristae, and dose-dependent increase of small fatty droplets. These microsteatotic hepatocytes were distributed mainly in the pericentral area. At higher cereulide doses, massive degeneration of hepatocytes occurred. The serum values of hepatic enzymes were highest on days 2-3 after the inoculation of cereulide, and rapidly decreased thereafter. General recovery from the pathological changes and regeneration of hepatocytes was observed after 4 weeks.     

Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae

An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described. The first phase of the fermentation is a batch phase with minimum generation times (1.4 h). During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained. Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely. A maximum cell concentration of 2.2 × 10⁷ cells/ml and a dry weight of 54 g/l have been obtained. As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates. In addition, the cells remained viable for several weeks. Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes. By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes. However, even a T. pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks. Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficieny of the system.Correspondence to: A. Tiedtke     

Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.