Affinity chromatography of an ethylene-synthesizing enzyme from red alga Porphyra tenera on an immobilized inhibitor of ethylene evolution

A method was established to purify acrylate decarboxylase fromPorphyra tenera by affinity chromatography using a proteinaceousinhibitor of ethylene evolution in marine algae, isolated fromP. tenera as a ligand. The proteinaceous inhibitor was covalentlycoupled to porous glass via four different spacer arms. Theporous glass-aminopropyltriethoxysilane-succinatephenylendiamine-succinate-inhibitorappeared to be the best derivative for retaining acrylate decarboxylaseextracted from P. tenera. Acrylate decarboxylase was extracted from 10 kg of P. teneraand semi-purified by ammonium sulfate. preparation and gel filtrationon Sephadex G-100. The active fraction was applied to an affinitycolumn. Acrylate decarboxylase was eluted in the starting buffercontaining 0.2 M NaCl. Ethylene formation from acrylate wasdetected in the presence of this enzyme extract, but not inthe case of the boiled enzyme extract. Acrylate decarboxylasewas inhibited by the inhibitor isolated from P. tenera. Thesefacts indicate that the formation of ethylene in marine algaefrom acrylate proceeds enzymatically. ² Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Ibaraki 300-21,Japan. (Received July 13, 1976; )     

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.     

Isolation and Characterization of a Mo6+ -Reducing Bacterium

A Mo⁶⁺ -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo⁶⁺, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo⁶⁺ (10 mM), the bacterium reduced Mo⁶⁺ to form molybdenum blue. Approximately 27% of Mo⁶⁺ added to the medium was reduced after 28 h of cultivation. The reduction of Mo⁶⁺ with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo⁶⁺ reduction. NADH and N,N,N′,N′ -tetramethyl-p-phenylenediamine served as electron donors for Mo⁶⁺ reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo⁶⁺ reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo⁶⁺ reduction. Both ferric and stannous ions strongly enhanced the activity of Mo⁶⁺ reduction by NADH.     

Endomucin is expressed in embryonic dorsal aorta and is able to inhibit cell adhesion

Glucagon-like peptide-1 (GLP-1) is an incretin, which induces glucose-dependent insulin secretion. GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. We investigated whether DPPIV-deficient F344/DuCrj rats show improved glucose tolerance when compared with DPPIV-positive F344/Jcl rats. Oral glucose tolerance test indicated improved glucose tolerance in F344/DuCrj rats, but blood glucose levels of the two strains were almost the same 120 min after the glucose bolus. Valine-pyrrolidide, a DPPIV inhibitor, had no effect on the glucose tolerance of F344/DuCrj rats, but improved that of F344/Jcl rats. Enhanced insulin secretion and high plasma active GLP-1 levels were detected in an intraduodenal glucose tolerance test. Glucose tolerance is improved in DPPIV-deficient F344/DuCrj rats via enhanced insulin release mediated by high active GLP-1 levels. Our results suggest that DPPIV inhibition is a rational strategy to treat diabetic patients by improving glucose tolerance with low risk of hypoglycemia.     

T cell receptor-mediated signaling induces GRP78 expression in T cells: the implications in maintaining T cell viability

The 78-kDa glucose-regulated protein (GRP78) is an important molecular chaperone in the endoplasmic reticulum (ER) induced by various stresses. This study showed that stimulation with anti-CD3 mAb, PMA plus ionomycin, or an antigen increased the levels of GRP78 mRNA in primary T cells, which was inhibited by Ca²⁺ chelators EGTA and BAPTA-AM and by an inhibitor of calcineurin FK506. In addition, the specific knockdown of GRP78 protein expression induced apoptosis in mouse EL-4 T cell line associated with CHOP induction and caspase-3 activation. Furthermore, overexpression of GRP78 inhibited PMA/ionomycin-induced cell death in EL-4 cells. Collectively, GRP78 expression is induced by TCR activation via a Ca²⁺-dependent pathway and may play a critical role in maintaining T cell viability in the steady and TCR-activated states. These results suggest a novel regulatory mechanism and an essential function of GRP78 in T cells.     

Confirmation of clonal reproduction of Fagus crenata Blume from Sado Island,Niigata Prefecture

While clonal growth is important in the East Asian Fagus subgenus Engleriana and the North American Fagus grandifolia (subgenus Fagus), for other subgenus Fagus species the vast majority of regeneration involves sexual reproduction with clonal growth only rarely observed. Here we aim to confirm using nuclear microsatellite markers whether clonal growth occurs in the Japanese endemic species Fagus crenata (subgenus Fagus) by investigating the origin of multi-stemmed clumps found within a high-elevation dwarf beech forest on Sado Island, Niigata Prefecture. We found that all stems collected from three separate clumps belonged to the same clump-specific multi-locus genotypes. The maximum size of clones was 3–4 m in diameter, comparable in size to those seen in the predominantly asexually regenerating Japanese species Fagus japonica (subgenus Engleriana). The species capacity for clonal growth is likely to underlie its ability to persist at high-elevation exposed sites at the limits of its ecological range.     

The effect of ATP and acetylcholine on the sodium transport in bullfrog large intestine (author's transl)]

By Ussing's flux chamber method the effect of ATP and acetylcholine (ACh) on the sodium transport was studied in bullfrog colon. The results obtained are as follows; 1. ATP added to the mucosal medium caused biphasic changes in the transmural potential difference (P.D.) and short-circuit current (S.C.C.), although serosal ATP was ineffective. After an initial rapid and transient rise, both the P.D. and S.C.C. increased in parallel to reach a peak in about 10 min suggesting that the tissue conductance is little affected by ATP. Addition of ouabain to the serosal fluid depressed both the P.D. and S.C.C. and abolished the electrical responses to ATP. The application of ouabain to the mucosal side did not cause any significant depression. These results can be explained in terms of stimulation of sodium pump by ATP added to the mucosal medium. 2. ACh added to either the mucosal or the serosal medium caused increased in the P.D. and the S.C.C. The serosal application was more effective than the mucosal application. The increase in S.C.C. was more remarkable than that in the P.D., indicating an increase in the tissue conductance. It is suggested that ACh stimulates ion transport systems by changing the membrane permeability of the colon.