Effects of Ca2+ and sulfhydryl reductant on the polymerization of soybean glycinin catalyzed by mammalian and microbial transglutaminases

Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.     

Very low vitamin C activity of orally administered L-dehydroascorbic acid.

The biological activity of L-dehydroascorbic acid (DHA), which is easily formed from L-ascorbic acid (ASC) during storage and cooking processes, has been considered to be equivalent to that of ASC on the basis of studies made several decades ago, when a specific method to determine ASC was not available. The nutritional activity of orally ingested DHA has now been evaluated by comparing ASC concentrations in 12 tissues of rats administered four different doses of ASC. Determinations were made by using the specific and sensitive method, which had been developed by us. Here it is shown that the efficiency of DHA was almost 10% of that of ASC on a molar basis, based on animal experiments using the inherently scorbutic ODS rat, which is a convenient human model animal to investigate the metabolism of vitamin C. On the basis of these findings, it is proposed that it is necessary to reevaluate the nutritional requirement of vitamin C based on both ASC and DHA contents of foods.     

Hepatic AA amyloidosis in a cat: cytologic and histologic identification of AA amyloid in macrophages

A 3‐year‐old, spayed female, Domestic Shorthair cat presented with anorexia, lethargy, vomiting, probable hemoabdomen, and multiple masses on the right lateral liver lobe. Clinicopathologic and imaging abnormalities included anemia, azotemia, icterus, and hepatomegaly with hypoechoic masses. On cytologic evaluation of a fine‐needle aspiration of a liver mass there was abundant extracellular pink‐ to purple‐colored material between hepatocytes. The amorphous material was stained with direct fast scarlet (DFS), and green birefringent areas were observed under polarized light, confirming the presence of amyloid. A unique finding on the cytologic smear were macrophages containing amorphous and fibrillar amyloid‐like protein. Histopathologic examination using H&E and Congo red staining confirmed amyloid deposits within the space of Disse, along the sinusoids, portal tracts, blood vessel walls, and within the cytoplasm of macrophages. Immunohistochemical staining with anti‐AA amyloid antibodies further confirmed the presence of AA amyloid. To the author's knowledge, this is the first report of the cytologic finding of AA amyloid protein within macrophages and DFS stain detection of amyloid on a cytologic smear.     

Molecular survey of Babesia infection in dogs in Okinawa, Japan

A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.     

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.     

Improvement of the functional properties of sucrose stearate by phosphorylation

Phosphorylated sucrose stearate (SE-P) was prepared by dry-heating sucrose stearate (SE) with metaphosphoric acid. The main product was deduced to be a monophosphosucrose monostearate by chemical analysis and mass spectrometry. SE-P exhibited remarkably higher solubility and emulsifying properties than SE, especially in the acidic region and in the presence of NaCl, and SE-P bound Ca2+ at a 1:1 molar ratio (SE-P/Ca2+). SE-P markedly reduced the viscosity of potato starch paste and inhibited retrogradation, whereas SE did not reduce it so much. It is thus expected that phosphorylation would be an appropriate method for improving the functional properties of SE and that SE-P could be used as a novel emulsifier and modifier with Ca2+-binding ability for starchy foods.     

Procyanidin-rich extract from grape seeds prevents cataract formation in hereditary cataractous (ICR/f) rats

Antioxidants such as vitamin C, vitamin E, and carotenoids have been reported to prevent the progression of experimentally induced cataracts. However, little is known of the effect of procyanidins, a powerful antioxidant, on cataract formation. This paper investigates the anticataract activity of grape seed extract (GSE, which contains 38.5% procyanidins) in hereditary cataractous rats (ICR/f rats). The ICR/f rats were fed a standard diet containing 0 or 0.213% GSE [0.082% procyanidins in the diet (w/w)] for 27 days. The GSE significantly prevented and postponed development of cataract formation by evaluation of slit lamp observations of the rats' eyes. Lens weight and malondialdehyde concentration in the lens and plasma cholesteryl ester hydroperoxide (ChE-OOH) level induced by CuSO4 were significantly lower in the GSE group compared with the control group. The rats were also fed for 14 days either the diet containing 0.085% procyanidin dimer to tetramer fraction (0.085% as the procyanidins), the diet containing 0.090% procyanidin pentamer to heptamer fraction (0.085% as the procyanidins), or the diet containing 0.093% procyanidin oligomers more than decamer fraction (0.085% as the procyanidins). The ChE-OOH levels in the procyanidin pentamer to heptamer and procyanidin oligomers more than decamer groups were significantly lower than in the procyanidin dimer to tetramer group. These results suggested that procyanidins and their antioxidative metabolites prevented the progression of cataract formation by their antioxidative action. The larger molecular procyanidins in the GSE might contribute this anticataract activity.     

Forces for morphogenesis investigated with laser microsurgery and quantitative modeling

We investigated the forces that connect the genetic program of development to morphogenesis in Drosophila. We focused on dorsal closure, a powerful model system for development and wound healing. We found that the bulk of progress toward closure is driven by contractility in supracellular "purse strings" and in the amnioserosa, whereas adhesion-mediated zipping coordinates the forces produced by the purse strings and is essential only for the end stages. We applied quantitative modeling to show that these forces, generated in distinct cells, are coordinated in space and synchronized in time. Modeling of wild-type and mutant phenotypes is predictive; although closure in myospheroid mutants ultimately fails when the cell sheets rip themselves apart, our analysis indicates that beta(PS) integrin has an earlier, important role in zipping.     

EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.