Amino acid intrinsic alpha-helical propensities III: positional dependence at several positions of C terminus

In this study, we have analyzed experimentally the helical intrinsic propensities of non-charged and non-aromatic residues at different C-terminal positions (C1, C2, C3) of an Ala-based peptide. The effect was found to be complex, resulting in extra stabilization or destabilization, depending on guest amino acid and position under consideration. Polar (Ser, Thr, Cys, Asn, and Gln) amino acids and Gly were found to have significantly larger helical propensities at several C-terminal positions compared with the alpha-helix center (-1.0 kcal/mole in some cases). Some of the nonpolar residues, especially beta-branched ones (Val and Ile) are significantly more favorable at position C3 (-0.3 to -0.4 kcal/mole), although having minor differences at other C-terminal positions compared with the alpha-helix center. Leu has moderate (-0.1 to -0.2 kcal/mole) stabilization effects at position C2 and C3, whereas being relatively neutral at C1. Finally, Met was found to be unfavorable at C1 and C2 ( +0.2 kcal/mole) and favorable at C3 (-0.2 kcal/mole). Thus, significant differences found between the intrinsic helical propensities at the C-terminal positions and those in the alpha-helix center must be accounted for in helix/coil transition theories and in protein design.     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.     

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.     

NADPH-diaphorase and calcium binding proteins in the trigeminal nucleus oralis of rats

We have examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV), in the trigeminal nucleus oralis (Sp5O). NADPH-d was detected by histochemistry while CBP was detected by immunohistochemistry. NADPH-d-positive neurons were distributed in the medial rostro-dorsomedial part (RDMsp5O) and dorsomedial part (DMsp5O) of Sp5O, and the rostrolateral part of the nucleus of the solitary tract (NTS). CB- and CR-positive neurons were mainly distributed in the dorsal part of Sp5O. In contrast, PV-positive neurons were mainly distributed in the ventral part of Sp5O. NADPH-d colocalized with CB (40%) and CR (20%) but not with PV in neurons of DMsp5O/ NTS. The mean cell sizes of neurons in RDMsp5O were larger than those in DMsp5O/NTS. PV-positive neurons were larger than NADPH-d-positive neurons. NADPH-d-, CB- and CR-positive neurons were generally small in RDMsp5O and DMsp5O/NTS. Few neurons were retrogradely labeled in RDMsp5O and DMsp5O from the thalamus, when numerous labeled neurons were in the principal and interpolar nuclei. These data indicate that NADPH-d histochemistry and CB, CR and PV immunohistochemistry identify a discrete cell population in Sp5O. Those labeled neurons in RDMsp5O and DMsp5O/NTS were considered to be involved in sensorimotor reflexive function of the intra-oral structures.     

Importance of permafrost as a source of water for plants in east Siberian taiga

Stable oxygen isotope ratios of plant water (sap water) were observed at Spasskaya Pad experimental forest near Yakutsk, Russia in 1997–1999. The ¹⁸O of sap water in larch trees (Larix gmelinii) decreased soon after leaf unfolding every year, indicating that snowmelt water was used in the beginning of summer. During mid to late summer, a clear difference in the water source used by plants was observed between wet summers and severe drought summers. The ¹⁸O values of water in larch trees were high (–17.8 to –16.1) in August 1999 (wet summer), but low (–20.4 to –19.7) in August 1998 (drought summer). These results indicated that plants used rainwater during a wet summer, but meltwater from permafrost was used by plants during a drought summer. One important role of permafrost is to provide a direct source of water for plants in a severe drought summer; another role is to keep surplus water in the soil until the next summer. If this permafrost system is disturbed by future global warming, unique monotypic stands of deciduous larch trees in east Siberia might be seriously damaged in a severe drought summer.     

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.