Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.     

Isolation of three main sericin components from the cocoon of the silkworm,Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.     

Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI)

The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.     

Probing the structural dynamics of nucleic acids by quantitative time-resolved and equilibrium hydroxyl radical "footprinting"

For many years, hydroxyl radical footprinting has been an insightful probe of the solvent accessibility of local regions of nucleic acid structure. Recently, quantitative applications of this technique have been developed that allow time-resolved and equilibrium analysis of transitions involving nucleic acid ligand binding and conformation change to be analyzed incisively.     

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

Albutensin A and complement C3a decrease food intake in mice

Albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg) derived from serum albumin dose-dependently decreased food intake after intracerebroventricular (10-50 nmol/mouse) or peripheral (0.3-1.0 micromol/mouse) administration in fasted conscious ddY mice. Albutensin A delayed gastric emptying and elevated blood glucose levels. Although albutensin A showed low affinity for bombesin receptor, it decreased food intake in bombesin receptor knockout mice, indicating that its inhibitory effect on feeding was not mediated through bombesin receptor. Then, we investigated whether the albutensin A-induced decrease in food intake was mediated by complement C3a and C5a receptors, because albutensin A had affinities for these receptors. Des-Arg-albutensin A, lacking affinity for C3a and C5a receptors, did not inhibit food intake. We found for the first time that centrally administered C3a (10-100 pmol/mouse) by itself decreased food intake in fasted mice. In contrast, C5a increased food intake after central injection. Based on these results, we conclude that the inhibitory effect of albutensin A on food intake is mediated through the C3a receptor.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.