Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation

Spermatogenesis is dependent on a small population of stem cells. Although stem cells are believed to expand infinitely, there is little functional evidence regarding whether spermatogonial stem cells can increase in their number. Using the spermatogonial transplantation technique, we evaluated the proliferative potential of spermatogonial stem cells in two models of regeneration. After busulfan injection to deplete stem cells, the surviving stem cells were able to expand by at least 15.8-fold within 2 mo. On the other hand, a serial transplantation study indicated that one transplanted stem cell was able to expand by 3.8- and 12-fold within 2 and 4 mo, respectively. These results provide direct functional evidence for the expansion of stem cells and establish the basis for further characterization of the stem cell self-renewal process.     

Piperine analogs in a hydrophobic fraction from Piper ribersoides (Piperaceae) and its insect antifeedant activity

Piper ribersoides Wall. (Piperaceae), which is called “Khua Sa khan” in the local language, is mainly grown in Laos. This plant is used as a food in Laos, but no report on its metabolites exists. Crushed stems were immersed in methanol. The ethyl acetate fraction showed potential insect antifeedant activity for Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and some of the active compounds were piperine analogs. Piperine and its geometrical isomer independently showed potent antifeedant activities, and piperine was presumably the main active compound in the methanol extract. Interestingly, we discovered that the antifeedant activity was reduced when they were mixed (50:50).     

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat

Abstract

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.     

Conserved Pyridoxal Protein That Regulates Ile and Val Metabolism

Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5′-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggS-deficient E. coli strain (ΔyggS) and a purified form of YggS, respectively. In the stationary phase, the ΔyggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of l-Val in the culture medium. The log-phase ΔyggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, l-Val. It also exhibited a 1.3- to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ΔyggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ΔyggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ΔyggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Forest restoration following a windthrow: how legacy retention versus plantation after salvaging alters the trajectory of initial recovery

Recently, the conversion of plantations into natural forests has attracted increasing attention, but little is known about how to convert a windthrown plantation into a natural forest. We examined the initial 3-year effects of salvage logging, site preparation, planting, and weeding in comparison with the effects of legacy retention after a typhoon blowdown on the changes in terrestrial environments and plant compositions and the similarity of the species composition to that of the intact natural forest. Our major findings are as follows: (1) A homogeneous, sunny, and flat environment continued for 3 years in the planted and weeded plot, whereas a heterogeneous environment was observed in the legacy retention plot; however, the light environment changed slightly in the planted plot. (2) The species composition in the planted plots changed greatly due to invasion by annual, biennial, and perennial herbs and early successional trees. Alien species also invaded the planted row. Conversely, the species composition changed little in the legacy retention, which was likely due to the presence of residual plants that prevented shade-intolerant species from being established. (3) The legacy retention, followed by the residual row, was the closest to the natural forest with respect to species composition, whereas the planted row was the most dissimilar from the natural forest. Legacy retention appears to be the most appropriate way to restore a natural forest immediately after a windthrow, yet supplemental planting may be necessary to restore the desired broadleaved species.     

Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease

Cerebral amyloid angiopathy (CAA) due to beta-amyloid (Abeta) is one of the specific pathological features of familial Alzheimer's disease. Abeta mainly consisting of 40- and 42-mer peptides (Abeta40 and Abeta42) exhibits neurotoxicity and aggregative abilities. All of the variants of Abeta40 and Abeta42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the Abeta40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type Abeta40. Similar tendency was observed for Abeta42 mutants at positions 22 and 23 whose neurotoxicity was 50-200 times stronger than that of the corresponding Abeta40 mutants, suggesting that these Abeta42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-Abeta42 and D23N-Abeta42 was similar to that of wild-type Abeta42, E22Q-Abeta42 and E22K-Abeta42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of Abeta mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that beta-sheet content of the Abeta mutants correlates with their aggregation. However, beta-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue beta-turn significantly enhanced the aggregative ability.