Studies on Phyto-peroxidase

In a continuation of the structural studies on Japanese-radish peroxidase a. the products resulting from the action of pepsin on performic acid-oxidized apo-peroxidase a have been examined by ion-exchange chromatography on a Dowex 50W-X2 column, followed by gelfiltration chromatography on a Sephadex G-25 column and by high voltage paper electrophoresis. Seven peptides have been isolated in purified forms in yields of 6 to 39 per cent, and their amino acid compositions have been determined.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

The Cell End Marker Protein TeaC Is Involved in Growth Directionality and Septation in Aspergillus nidulans

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In Schizosaccharomyces pombe it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to Aspergillus nidulans. Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of teaC affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of teaA deletion (tea1) strains. Some small, anucleate compartments were observed. Overexpression of teaC repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.Filamentous fungi represent fascinating model organisms for studying the establishment and maintenance of cell polarity, because cell growth takes place at the tip of the extremely elongated hyphae. Hyphal extension requires the continuous expansion of the membrane and the cell wall and is driven by continuous fusion of secretion vesicles at the tip (8, 12). The transportation of vesicles is probably achieved by the coordinated action of the MT and the actin cytoskeleton. According to one model, vesicles first travel along MTs, are unloaded close to the hyphal tip, where they form a microscopically visible structure the “Spitzenkörper,” which is also called the “vesicle supply center,” referring to the assumed function (24, 25). For the last step, vesicle transportation from the Spitzenkörper to the apical membrane, actin-myosin-dependent movement is used. Anti-cytoskeletal drug experiments have shown that hyphae can grow for some time in the absence of MTs but not in the absence of the actin cytoskeleton (14, 27, 30a).In Schizosaccharomyces pombe it was shown clearly that the polarization of the actin cytoskeleton depends on the MT cytoskeleton (2, 7). In 1994, polarity mutants of S. pombe were isolated and subsequent cloning of one of the genes identified the polarity determinant Tea1 (19, 29). Because this protein labels the growing cell end, this and other subsequently isolated proteins of this class were named cell end markers. It was shown that cell end localization of Tea1 requires the activity of a kinesin motor protein, Tea2, which transports the protein to the MT plus end (3). Together with the growing MT, Tea1 reaches the cortex, where it is unloaded and binds to a prenylated and membrane-anchored receptor protein, Mod5 (28). The formin For3, which catalyzes actin cable formation, is recruited to the tip through binding to another cell end marker protein, Tea4, which confers tethering to Tea1 (7, 18, 33). Tea4 is required for For3 localization at the cell tip, specifically during initiation of bipolar growth (18).Recently, it was shown that components of this polarity determination machinery are conserved in the filamentous fungus A. nidulans (8). The first component identified was the Tea2 homologue, KipA, a kinesin-7 motor protein (16). Deletion of the gene did not affect hyphal tip extension but polarity determination. Instead of growing straight, hyphae grew in curves. KipA moves along MTs and accumulates at the MT plus end. The identification of Tea1 and a Mod5 homologue was more difficult, because the primary structure of these cell end marker proteins is not well conserved in filamentous fungi. A Tea1 homologue, TeaA, only displayed 27% sequence identity. However, the presence of Kelch repeats in both proteins suggested conserved functions (31). A Mod5 homologue was identified by a conserved CAAX prenylation motif at the C terminus. Systematic analyses of proteins with such a motif in the A. nidulans genome led to the identification of TeaR. Like Tea1 and Mod5, TeaA and TeaR localize at or close to the hyphal membrane at the growing cell end (31). However, correct localization of TeaR requires TeaA. In addition, sterol-rich membrane domains define the place of TeaR attachment to the hyphal tip. In contrast to S. pombe, TeaA and TeaR are still transported to the hyphal tip in the absence of the motor protein KipA, but their localization is disturbed in comparison to wild type. This suggests that other proteins are necessary for exact TeaA positioning, whose localization depends on KipA.We characterized a homologue of the S. pombe cell end marker protein, Tea4, and found that the protein is required for the maintenance of straight polar growth but that it also appears to be involved in septation.     

Salinity decreases nitrite reductase gene diversity in denitrifying bacteria of wastewater treatment systems

Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant nirK clone was related to nirK of the genus ALCALIGENES:     

Reduction of E-cadherin by human defensin-5 in esophageal squamous cells

Barrett’s esophagus (BE) is metaplastic columnar epithelium converted from normal squamous epithelia in the distal esophagus that is thought to be a precancerous lesion of esophageal adenocarcinoma. BE is attributed to gastroesophageal reflux disease (GERD), and therefore gastric acid or bile acids are thought to be factors that cause epithelial cell damage and inflammation in the gastro-esophageal junction. The decrease of adherent junction molecules, E-cadherin has been reported to be associated with the progression of the Barrett’s carcinoma, but the initiation of BE is not sufficiently understood. BE is characterized by the presence of goblet cells and occasionally Paneth cells are observed at the base of the crypts. The Paneth cells possess dense granules, in which human antimicrobial peptide human defensin-5 (HD-5) are stored and secreted out of the cells. This study determined the roles of HD-5 produced from metaplastic Paneth cells against adjacent to squamous cells in the gastro-esophageal junction. A human squamous cell line Het-1A, was incubated with the synthetic HD-5 peptide as a model of squamous cell in the gastro-esophageal junctions, and alterations of E-cadherin were investigated. Immunocytochemistry, flowcytometry, and Western blotting showed that the expression of E-cadherin protein was decreased. And a partial recovery from the decrease was observed by treatment with a CD10/neprilysin inhibitor (thiorphan). In conclusion, E-cadherin expression in squamous cells was reduced by HD-5 using in vitro experiments. In gastro-esophageal junction, HD-5 produced from metaplastic Paneth cells may therefore accelerate the initiation of BE.     

Studies on Rice Glutelin

The complete amino acid analysis of the whole glutelin preparation from rice endosperm was performed. The recoveries were 101.59% for amino acid residues and 101.68% for nitrogen, and the standard deviations for four determinations on the 22 and 70 hr hydrolyzates were very small. The features of the amino acid composition of the protein were as follows; (1) the high contents of dicarboxylic amino acids, particularly glutamic acid, (2) about 60% of these dicarboxylic amino acids was in the amide form, and (3) the significantly low contents of tryptophan, methionine and half cystine. The amino acid analyses of the two kinds of the subunits of glutelin, the neutral major one and the basic minor one, were also carried out. There were some significant differences between the two subunits, for instance, in the contents of glutamic acid, tryptophan, glycine, half cystine, methionine and lysine. However, the composition of whole glutelin seemed to be settled predominantly by that of the major subunit.     

A cell wall protein (YqgA) is genetically related to the cell wall-degrading dl-endopeptidases in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has a thick cell wall. The cell wall contains various proteins, both for secretion and for peptidoglycan (PG) maintenance. Penicillin-binding proteins for PG synthesis, PG hydrolases (autolysins), and regulator proteins for the autolysins are the known components of the PG maintenance system. YqgA was identified as an abundant protein attached to the cell wall of B. subtilis through a proteomics analysis. The YqgA protein was localized at cell division sites during the transition period between the exponential and the stationary phases. YqgA localization was affected by mutations in the dl-endopeptidases (DLEPases), which are the autolysins involved in cell morphogenesis. Furthermore, yqgA mutations on a background of defective DLEPases led to delays in cell growth and cell morphological changes. These results demonstrate that yqgA is genetically related to the genes encoding DLEPases involved in cell morphogenesis.     

Denatured human alpha-defensin attenuates the bactericidal activity and the stability against enzymatic digestion

alpha-Defensin is an antimicrobial peptide which plays an important role in innate immunity. Human defensin (HD)-5 is stored in the Paneth cells of the small intestine as a pro-form and is cleaved by trypsin, which is co-secreted from the Paneth cell granules. The mature HD-5 is protected from further digestion by the proteolysis enzyme. We generated both recombinant HD-5 and proHD-5, and the reduced form of each peptide in order to determine their physiological roles of the disulfide bonds. The reduced proHD-5 attenuated the bactericidal activity and the stability against the trypsin digestion. Human defensin was protected from the enzymatic degradation by disulfide bridges. We further purified the HD-5 with a disulfide variation in the small intestine of Crohn's disease patients. The HD-5 was sensitive to the trypsin treatment. These observations evidently predict that a defensin deficiency may be caused by a disulfide disorder in the disease.