Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.     

Nucleotide changes in the translated region of SCN5A from Japanese patients with Brugada syndrome and control subjects

The mutations of the SCN5A gene have been implicated to play a pathogenetic role in Brugada syndrome, which causes ventricular fibrillation. To determine the Brugada-associated mutations in Japanese patients, facilitate pre-symptomatic diagnosis, and allow genotype-phenotype studies, we screened unrelated patients with Brugada syndrome for mutations. DNAs from 6 Japanese patients were obtained and the sequence in the translated region of SCN5A was determined. We could not find the mutations reported previously, but found 17 sites of nucleotide change, consisting of 7 synonymous and 10 non-synonymous nucleotide changes in our patients. Among them, two non-synonymous nucleotide changes (G1663A and G5227A) are specific to our patients and these changes were not found in 53 healthy controls. In 4 patients out of 6, no specific nucleotide change for Brugada syndrome could be detected. Our findings demonstrating no patient-specific change in the translated region of the SCN5A gene among two thirds of the small number of patients examined here imply that another gene other than the SCN5A may be associated with this disease, supporting previous investigations in Japan and other countries.     

Theanine,gamma-glutamylethylamide,is metabolized by renal phosphate-independent glutaminase

The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response. Recently, we have demonstrated that treatment of tobacco ( Nicotiana tabacum ) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li⁺, Na⁺, K⁺), alkali earth metals (Mg²⁺, Ca²⁺) or lanthanides (La³⁺, Gd³⁺). In this study, we tested the effect of extracellular supplementation of Zn²⁺ and Mn²⁺ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent. Extracellular supplementation of Zn²⁺ and Mn²⁺ inhibited the generation of superoxide in response to addition of salts. Although both Zn²⁺ and Mn²⁺ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn²⁺ simply inhibited total production of superoxide while Zn²⁺ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn²⁺ and Zn²⁺ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed.     

Spectroscopic evidence that salicylic acid converts a temporally inactivated form of horseradish peroxidase (compound III) to the irreversibly inactivated verdohemoprotein (P-670)

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-catalyzed reactions are discussed.     

Dietary diacylglycerol suppresses high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats

We have recently shown that the long-term ingestion of dietary diacylglycerol (DAG) mainly containing 1,3-isoform reduces body fat accumulation in humans as compared to triacylglycerol (TAG) with the same fatty acid composition. The fat reduction in this human experiment was most pronounced in visceral fat and hepatic fat. Recent animal studies have also indicated that dietary DAG induces alteration of lipid metabolism in the rat liver. In the present study, the dietary effects of DAG on high fat diet-induced hepatic fat accumulation and hepatic microsomal triglyceride transfer protein (MTP) activity were examined in comparison with those of TAG diet in rats. When the TAG oil content was increased from 10 to 30 g/100 g diet, hepatic TAG concentration, hepatic MTP activity and MTP large subunit mRNA levels were significantly increased after 21 days. However, when the dietary TAG oil (30 g/100 g diet) was replaced with the same concentration of DAG oil with the same fatty acid composition, the increase of the TAG concentration and the MTP activity in the liver were significantly less and the mRNA levels remained unchanged. The MTP activity levels correlated significantly with hepatic TAG concentration.These results showed that dietary DAG may suppress high fat diet-induced MTP activity in the liver, and indicated the possibility that hepatic TAG concentration may regulate hepatic MTP activity.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.