Important role of methionine 145 in dimerization of bovine β-lactoglobulin

β-Lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.     

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.     

Sodium orthovanadate suppresses DNA damage-induced caspase activation and apoptosis by inactivating p53

We previously reported that p42/SETbeta is a substrate for caspase-7 in irradiated MOLT-4 cells, and that treating the cells with sodium orthovanadate (vanadate) inhibits p42/SETbeta's caspase-mediated cleavage. Here, we initially found that the inhibitory effect of vanadate was due to the suppression of caspase activation but not of caspase activity. Further investigations revealed that vanadate suppressed upstream of apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax, and p53 transactivation, although the accumulation, total phosphorylation, and phosphorylation of six individual sites of p53 were not affected. Importantly, vanadate suppressed p53-dependent apoptosis, but not p53-independent apoptosis. Finally, gel-shift and chromatin immunoprecipitation assays conclusively demonstrated that vanadate inhibits the DNA-binding activity of p53. Vanadate is conventionally used as an inhibitor of protein tyrosine phosphatases (PTPs); however, we recommend that the influence of vanadate not only on PTPs but also on p53 be considered before using it.     

Inhibitory effect of combined administration with CPT-11 and 5-fluorouracil in vitro and in vivo

Combined antitumor activity of CPT-11 and 5-fluorouracil (5-FU) was evaluated in a human cultured cell line derived from lung cancer. After 24 h culture with SN-38 followed by 5-FU 24 h, synergistic effect was observed in the cell line. In addition, the antitumor effect of this combination was studied in in vivo experiments using Donryu rat with Yoshida sarcoma cells. CPT-11 and 5-FU synergistically inhibited tumor growth. There was no significant increase of toxicity as assessed by the body weights. These results might support for the combination with 5-FU and CPT-11 in a chemotherapy for cancer.     

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:     

Production of arachidonic acid by a hydrocarbon-utilizing strain of Penicillium cyaneum

Summary Penicillium cyaneum, isolated from an, oil-field, was grown in batch culture on media containing glucose or kerosene alone, or both glucose and kerosene. Arachidonic acid was produced in highest yield (7.5%; 200 g per g dry weight of organisms) in media containing glucose and kerosene or kerosene alone. No effect on the arachidonic acid yield was observed when either yeast extract or sodium nitrate was used as the nitrogen source. Higher yields of intracellular fatty acids and arachidonic acid were obtained at 27°C than at 32°C.