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.     

Spectral Properties of Acid Denaturation of Rat Liver Arginase

Circular dichroism spectra in the ultraviolet region, similar to the spectrum of the collagen helix, were regenerated when various grades of industrial gelatin were allowed to stand at 5°C in a concentration low enough that they did not form gels. Spectrum generation was decreased by an addition of salt or alcohol and by raising the temperature. Maximal regeneration took place at the respective iso-ionic points of the acid- and alkali-processed gelatins. As judged from these spectra, 60% of the helical structure of collagen was regenerated from the industrial gelatin that had the highest α-chain content.     

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

Graphical classification of DNA sequences of HLA alleles by deep learning

Alleles of human leukocyte antigen (HLA)-A DNAs are classified and expressed graphically by using artificial intelligence “Deep Learning (Stacked autoencoder)”. Nucleotide sequence data corresponding to the length of 822 bp, collected from the Immuno Polymorphism Database, were compressed to 2-dimensional representation and were plotted. Profiles of the two-dimensional plots indicate that the alleles can be classified as clusters are formed. The two-dimensional plot of HLA-A DNAs gives a clear outlook for characterizing the various alleles.     

Study of the structure-activity relationship of polymyxin analogues

A structure-activity relationship study on three classes of polymyxin analogues focusing on hydrophobicity was conducted.     

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.     

Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains)

We screened a cDNA library of germinating rice seeds with a cDNA for aleurain (cysteine proteinase from barley) and obtained three distinct types of cDNA clones encoding three species of cysteine proteinases (oryzains alpha, beta, and gamma). The deduced amino acid sequences are distinct in part, but, on the whole, are similar to one another. The three sequences all contain the catalytic triad Cys25-His159-Asn175 (papain numbering). The three oryzains are similar to one another and also to other known cysteine proteinases such as papain and cathepsin H with respect to the sequences around the active site residues and the COOH-terminal Trp-rich region. Amino acid sequence comparison revealed that oryzains alpha and beta are similar not only to each other (70% similarity) but also to actinidin and papain (about 50%), whereas oryzain gamma was rather similar to aleurain (85%) and cathepsin H (60%). Northern blot analysis revealed that the mRNAs for the three oryzains are expressed only in seeds, not in shoots or roots, and show different expression profiles during germination and when the seeds are treated with gibberellic acid. Oryzains alpha and gamma are expressed continuously during germination with a maximum expression 5 days from the start of germination, but are present in neither ripening nor ripened seeds. On the other hand, oryzain beta is expressed not only during germination, but also in ripened seeds before germination. It was noted that the expression of the three oryzain mRNAs is enhanced in different manners by gibberellic acid but is not enhanced by other plant hormones such as auxin. The induction of oryzain beta mRNA is transient, reaching a maximum in 4 h from the addition of giberellic acid and diminishing rapidly thereafter, while the induction of oryzain alpha and gamma mRNAs continues over 5 days. Thus, multiple systems involving cysteine proteinases must be differentially involved in the germination process, probably under hormonal control.     

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.