Functional assembly of the Na+/H+ antiporter of Helicobacter pylori from partial fragments in vivo

Functional assembly of the Helicobacter pylori Na+/H+ antiporter (HPNhaA) from partial fragments was studied. Expression plasmids encoding a series of complementary N- and C-terminal fragment pairs containing the transmembrane domains (TMs) were constructed by inserting a stop or a start codon into each of the loop regions of NhaA. HPNhaA fragments alone or complementary fragment pairs were expressed in DeltanhaA Escherichia coli, and fragment integration into the membrane and antiporter activity were measured. TM1-10, TM1-11, TM2-12, TM6-12, and TM10-12 were found in the membrane fraction, while the other fragments were not. While no single fragment displayed antiporter activity, simultaneous expression of fragments in certain pairs, such as TM1-2 + TM3-12, TM1-8 + TM9-12, or TM1-11 + TM12, reconstituted antiporter activity. With the exception of TM12, all of the fragments in the pairs were detected in the membrane. No single fragments expressed alone for these pairs were found in the membrane, except for TM1-11, suggesting that the interaction between the fragments in these pairs stabilized the fragments and enabled reconstitution of HPNhaA. We also found that the simultaneous expression of three complementary fragments (TM1-2 + TM3-8 + TM9-12) reconstituted HPNhaA activity. Other pairs that were found in the membrane (TM1-5 + TM6-12, TM1-10 + TM11-12, and TM1 + TM2-12) did not reconstitute antiporter activity, suggesting that they may not have the proper conformation. These results revealed that the ability to reconstitute antiporter activity depends on the split position in the loop regions and the interaction between complementary fragment pairs. We propose that formation of the active HPNhaA molecule is initiated by the interaction of short-lived intermediates and maintained by the increased stability of the intermediates within the resulting complex.     

Metal binding ability of glutathione transferases conserved between two animal species, the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II)>V(IV)>Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Influence of calcium on proliferation and phenotype alteration of cardiomyocyte in vitro

An accelerated weight gain is noted in the heart of Ca-deficient, hypertensive chick embryos maintained in a shell-less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell-less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of (1) various Ca concentration in the culture medium (NCa, 1.8 mmol/L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and (2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle–specific and cardiomyocyte-specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1-S–specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)-specific, Ca-ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de-differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca-deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes. J. Cell. Physiol. 177:289–298, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and characterization of the sweet protein brazzein

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose, and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless. © 1996 John Wiley & Sons, Inc.     

Identification and characterization of HLA-B*5401-restricted HIV-1-Nef and Pol-specific CTL epitopes

The identification of HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by each HLA allele and the characterization of their CTL responses are important for the study of pathogenesis of AIDS and the development of a vaccine against it. In the present study, we focused on identification and characterization of HIV-1 epitopes presented by HLA-B*5401, which is frequently found in the Asian population, because these epitopes have not yet been reported. We identified these epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef. Seven of these 17-mer peptides induced HLA-B*5401-restricted CD8+ T cell responses. Only five HLA-B*5401-restricted Pol- or Nef-specific CD8+ T cell responses were detected in the analysis using 11-mer overlapping peptides. Three Pol and two Nef optimal peptides were identified by further analysis using truncated peptides. These epitope-specific CTLs effectively killed HLA-B*5401-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed by HLA-B*5401 in HIV-1-infected cells. These epitope-specific CD8+ T cells were elicited in more than 25% of chronically HIV-1-infected individuals carrying HLA-B*5401. Therefore, these epitopes should prove useful for studying the pathogenesis of AIDS in Asia and developing a vaccine against HIV-1.