Fo portion of Escherichia coli H+-ATPase. Carboxyl-terminal region of the b subunit is essential for assembly of functional Fo

Six chromosomal uncF mutants of Escherichia coli defective in the b subunit of H+-ATPase (156 amino acid residues) were identified (KF92, Met-1----Val; KF164, Gln-64----end; KF61 and KF144, Gln-104----end; KF138, Gln-106----end; and KF79, Gln-123----end). The membranes of all these mutants had low ATPase activities (less than 5% of that of the wild type), and no functional H+ pathway, although the truncated b subunits were integrated into these membranes. These findings suggest that about 30 carboxyl-terminal amino acid residues of the b subunit are essential for formation of the F1-binding site and H+ pathway. For examination of the role(s) of the carboxyl-terminal region(s) or residue(s) of the b subunit, recombinant plasmids carrying truncated uncF genes of various lengths were constructed by in vitro muta-genesis and introduced into a recA1 derivative of strain KF92 (Met-1----Val). Analyses of the membranes from the resulting strains demonstrated that almost the entire carboxyl-terminal region of the b subunit is necessary for formation of functional Fo, since loss of the carboxyl-terminal residue resulted in significant reduction of both F1 binding and H+ translocation, and loss of two or more residues abolished both activities completely.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport

The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.     

Overproduction of subunit a of the F0 component of proton-translocating ATPase inhibits growth of Escherichia coli cells.

A hybrid plasmid, pKY159, carrying the promoter and the proximal region of the gene cluster for proton-translocating ATPase caused growth inhibition of Escherichia coli cells (K. Yamaguchi and M. Yamaguchi, J. Bacteriol. 153:550-554, 1983). The mechanism of this growth inhibition was studied, especially in terms of the responsible gene(s). Insertion of IS1, IS5, or gamma delta between the promoter and the gene for a possible component of the ATPase of 14,000 daltons (14K protein) released the inhibitory effect by pKY159. Deletion of the gene for subunit a also released the effect. However, deletion in the gene for the 14K protein released the effect only with an additional insertion within the gene. These results suggested that overproduction of subunit a is closely related to growth inhibition, whereas the 14K protein is not.     

Convergence of Oropharyngolaryngeal, Baroreceptor and Chemoreceptor Afferents onto Insular Cortex Neurons in Rats

Forty-two neurons that responded to electrical stimulation ofat least one of four nerves, the chorda tympani (CT), the lingual-tonsillarbranch of the glossopharyngeal (LT-IXth) nerve, the pharyngealbranch of the glossopharyngeal (PH-IXth) nerve and the superiorlaryngeal (SL) nerve, were identified from the insular cortexby using glass microelectrodes in paralysed and anesthetizedrats. Four, 42, 41 and 40 neurons responded to the CT, LT-IXth,PH-IXth and SL nerve stimulation respectively. Of these 42 neurons,most (37/42, 88.1%) responded to three nerves (the LT-IXth,PH-IXth and SL), two (4.8%) responded to two nerves and theremaining three (7.1%) responded to all four nerves. No neuronsresponded to one specific stimulus. The responsiveness of these42 neurons to baroreceptor and chemoreceptor stimulation byan i.v. injection of three drugs was investigated. For baroreceptorstimulation, methoxamine hydrochloride (Mex) and sodium nitroprusside(SNP) were used; for chemoreceptor stimulation, sodium cyanide(NaCN) was used. Of the 42 neurons, 31 (73.8%) showed an excitatoryor inhibitory response to baroreceptor and chemoreceptor stimulationwith at least one of the three drugs, and the remaining 11 (26.2%)showed no response. Of these 31 baroreceptor and chemoreceptor-sensitiveneurons, 19(61.3%) responded to two or all three drugs, andthe rest (12; 38.7%) responded to one. Most neurons recordedwere distributed in the posterior insular cortex. These resultsindicate that the neurons in the posterior insular cortex receiveconvergent inputs from the oropharyngolaryngeal region, thebaroreceptors and the chemoreceptors, suggesting that the posteriorinsular cortex may integrate various sensory information. Chem.Senses 22: 399–406, 1997.     

Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies

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Apple S locus region represents a large cluster of related, polymorphic and pollen-specific F-box genes

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a complex S locus that encodes separate proteins for pistil and pollen specificities, extracellular ribonucleases (S-RNases) and F-box proteins SFB/SLF, respectively. SFB/SLFs of Prunus (subfamily Prunoideae of Rosaceae), Solanaceae and Plantaginaceae are single copy in each S haplotype, while recently identified pollen S candidates SFBBs of subfamily Maloideae of Rosaceae, apple and Japanese pear, are multiple; two and three related SFBBs were isolated from each S haplotype of apple and Japanese pear, respectively. Here, we show that apple (Malus × domestica) SFBBs constitute a gene family that is much larger than initially thought. Twenty additional SFBB-like genes/alleles were isolated by screening of a BAC library derived from S ³ S ⁹ genotype, and tentatively named MdFBX1-20. All but one MdFBX showed S haplotype-specific polymorphisms. All the polymorphic MdFBXs were completely linked to S-RNase in 239 segregants. In addition, FISH revealed that the monomorphic gene MdFBX11 is also located near S-RNase, and the S locus is located in a subtelomeric region of a chromosome and is not close to the centromere. All MdFBXs were specifically expressed in pollen, except for a pseudogene MdFBX4 that showed no expression in any organs analyzed. Phylogenetic analysis revealed that the closest relatives of most MdFBXs were from a different S haplotype, suggesting that proliferation of MdSFBB/FBXs predates diversification of the S haplotypes.     

Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.