No abstract

Oral colonization by Streptococcus gordonii, an important cause of subacute bacterial endocarditis, involves bacterial recognition of sialic acid-containing host receptors. The sialic acid-binding activity of this microorganism was previously detected by bacterium-mediated hemagglutination and associated with a streptococcal surface component identified as the Hs antigen. The gene for this antigen (hsa) has now been cloned in Escherichia coli, and its expression has been detected by colony immunoblotting with anti-Hs serum. Mutants of S. gordonii containing hsa inactivated by the insertion of an erythromycin resistance gene or deletion from the chromosome were negative for Hs-immunoreactivity, bacterium-mediated hemagglutinating activity, and adhesion to ␣2-3-linked sialoglycoconjugates. The deletion in the latter mutants was complemented by plasmid-borne hsa, resulting in Hs antigen production and the restoration of cell surface sialic acid-binding activity. The hsa gene encodes a 203-kDa protein with two serine-rich repetitive regions in its 2,178-amino-acid sequence. The first serine-rich region occurs within the amino-terminal region of the molecule, between different nonrepetitive sequences that may be associated with sialic acid binding. The second serine-rich region, which is much longer than the first, is highly repetitive, containing 113 dodecapeptide repeats with a consensus sequence of SASTSASVSASE. This long repetitive region is followed by a typical gram-positive cell wall anchoring region at the carboxyl-terminal end. Thus, the predicted properties of Hsa, which suggest an amino-terminal receptor-binding domain attached to the cell surface by a molecular stalk, are consistent with the identification of this protein as the sialic acid-binding adhesin of S. gordonii DL1.

In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 M BITC, characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane and caspase-3 activation, whereas no caspase activation and propidium iodide incorporation into cell were detected with 50 M BITC that induced necrosis. The mitochondrial death pathway was suggested to be involved in BITC-induced apoptosis because the treatment of cells with BITC-induced caspase-9-dependent apoptosis and mitochondrial transmembrane potential (⌬⌿m) alteration. We demonstrated here for the first time that BITC directly modifies mitochondrial functions, including inhibition of respiration, mitochondrial swelling, and release of cytochrome c. Moreover, glutathione depletion by diethyl maleate significantly accelerated BITC-triggered apoptosis, suggesting the involvement of a redox-dependent mechanism. This was also implicated by the observations that intracellular accumulation of reactive oxygen species, including superoxide (O 2 . ) and hydroperoxides (HPOs), was indeed detected in the cells treated with BITC and that the intracellular HPO level was significantly attenuated by pretreatment with N-acetylcysteine. The treatment with a pharmacological scavenger of O 2 . , Tiron, also diminished the HPO formation by ϳ80%, suggesting that most of the HPOs were H 2 O 2 derived from the dismutation of O 2 . . These results suggest that BITC induces apoptosis through a mitochondial redox-sensitive mechanism.

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of Streptococcus gordonii DL1 was previously associated with the hsa gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an hsa chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing hsa constructs encoding Hsa that lacked either the N-or C-terminal portion of SR2. In contrast, hsa constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of hsa. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction. Strains of Streptococcus gordonii, Streptococcus sanguinis,and Streptococcus oralis adhere to saliva-coated hydroxyapatite, an experimental model of the tooth surface, and also attach to host cells, including erythrocytes (RBC) and polymorphonuclear leukocytes (5,6,11,16,19). A common mechanism involved in these interactions is recognition of surfaceassociated host sialoglycoconjugates. Binding of S. gordonii DL1 to such receptors depends on the Hs antigen, a highmolecular-weight streptococcal surface component that appears to be glycosylated based on the reaction of this component with wheat germ agglutinin (WGA) and its labeling following periodate oxidation (25). The gene encoding this antigen, designated hsa, was identified by its expression in Escherichia coli by using anti-Hs antibody and was subsequently deleted from the S. gordonii DL1 chromosome (23). This deletion eliminated Hs antigen production and bacterial adhesion to immobilized sialic acid-containing receptors. Moreover, expression of hsa from a streptococcal plasmid restored these properties, firmly associating this gene with the sialic acid-binding adhesin of strain DL1 (23).The hsa gene encodes a large (203-kDa) serine-rich repeat protein composed of 2,178 amino acid residues. The predicted protein sequence includes an N-terminal nonrepetitive region ...

Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.

Gastric H؉ -ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K ؉ -ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.

Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes.