Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125.

Na+/H+ antiport was studied in alkaliphilic Bacillus sp. strain C-125, its alkali-sensitive mutant 38154, and a transformant (pALK2) with recovered alkaliphily. The transformed was able to maintain an intracellular pH (pHin) that was lower than that of external milieu and contained an electrogenic Na+/H+ antiporter driven only by delta psi (membrane potential, interior negative). The activity of this delta psi-dependent Na+/H+ antiporter was highly dependent on pHin, increasing with increasing pHin, and was found only in cells grown at alkaline pH. On the other hand, the alkali-sensitive mutant, which had lost the ability to grow above pH 9.5, lacked the delta psi-dependent Na+/H+ antiporter and showed defective regulation of pHin at the alkaline pH range. However, this mutant, like the parent strain, still required sodium ions for growth and for an amino acid transport system. Moreover, another Na+/H+ antiporter, driven by the imposed delta pH (pHin > extracellular pHout), was active in this mutant strain, showing that the previously reported delta pH-dependent antiport activity is probably separate from delta psi-dependent antiporter activity. The delta pH-dependent Na+/H+ antiporter was found in cells grown at either pH 7 or pH 9. This latter antiporter was reconstituted into liposomes by using a dilution method. When a transmembrane pH gradient was applied, downhill sodium efflux was accelerated, showing that the antiporter can be reconstituted into liposomes and still retain its activity.     

Alternate activity in the synergistic muscles during prolonged low-level contractions

The purpose ofthis study was to investigate the functional interrelationship betweensynergistic muscle activities during low-level fatiguing contractions.Six human subjects performed static and dynamic contractions at anankle joint angle of 110° plantar flexion and within the range of90-110° (anatomic position = 90°) under constant load(10% maximal voluntary contraction) for 210 min. Surfaceelectromyogram records from lateral gastrocnemius (LG), medialgastrocnemius (MG), and soleus (Sol) muscles showed high and silentactivities alternately in the three muscles and a complementary andalternate activity between muscles in the time course. In the secondhalf of all exercise times, the number of changes in activity increasedsignificantly (P < 0.05) in each muscle. The ratios of active to silent periods of electromyogram activity were significantly higher (P < 0.05) in MG (4.5 ± 2.2) and Sol (4.3 ± 2.8) than in the LG(0.4 ± 0.1), but no significant differences were observed betweenMG and Sol. These results suggest that the relativeactivation of synergistic motor pools are not constant during alow-level fatiguing task.

     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

TUCAN, an antiapoptotic caspase-associated recruitment domain family protein overexpressed in cancer.

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulated CARD-containing antagonist of caspase nine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation.     

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO₂Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO₂Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO₂Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO₂Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO₂Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Evaluation of nutritional availability and anti-tumor activity of selenium contained in selenium-enriched Kaiware radish sprouts

We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 microg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 microg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 mug Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 microg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.     

Purification and properties of cytochrome P-450 from homogenates of human fetal livers

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes.     

Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences

BACKGROUND: Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS: The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS: MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.