Lack of effect of carbohydrate depletion on some properties of human mast cell chymase

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This enzymatic deglycosylation product was enough to explore possibilities that N-glycan might modify some properties of human chymase. Substrate specificity, optimum pH and the elution profile from the heparin affinity gel were not affected by the deglycosylation. Only a slight but significant difference was observed in the Km value for conversion of angiotensin I to angiotensin II. Other kinetic constants such as kcat were not influenced. The kinetics of conversion of big endothelin-1 to endothelin-1(1-31) were not significantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline pH and thermal stress. Even at physiological temperature and pH, the activity of glycosylated human chymase was more stable. From these results, it appears that the N-glycan of human chymase contributes to the stability of this enzyme but not to its functional properties.     

Maximum production strategy for biodegradable copolymer P(HB-co-HV) in fed-batch culture of Alcaligenes eutrophus

A novel strategy for the maximum production of a biodegradable copolymer, poly(3-hydroxybutyric-co-hydroxyvaleric) acid, P(HB-co-HV), was developed, based on the kinetic parameters obtained from fed-batch culture experiments of Alcaligenes eutrophus. The effects of various culture conditions such as mole ratio of carbon:nitrogen in feed medium (C/N); total fatty acids concentrations; and addition ratio of fatty acids on cultivation properties such as the specific rates of cell formation, mu (h-1), P(HB-co-HV) production, rho[g.P(HB-co-HV)/g.cell/h], production yield from fatty acids [g.P(HB-co-HV)/g.fatty acid], and mole fraction of monomeric units in the copolymer [mol.(HV)/{mol.(HB) + mol.(HV)}], were investigated. When nitrogen supply was sufficient for cell growth; that is, C/N (mol.nitrogen atom/mol.carbon atom) was low, mu was high, but rho and the production yield were low, because fatty acids were used mainly for energy formation and anabolic reactions in the cells. On the other hand, when nitrogen supply was limited for cell growth-that is, C/N was high-rho was high. The highest value of rho was obtained when C/N was 75. As the mole ratio of valeric acid (VA) to butyric acid (BA) in the feed medium was increased, the mole fraction of HV units in P(HB-co-HV) increased linearly. When the ratio of BA to VA in the feed medium was kept at a constant value, but C/N was increased, the mole fraction of HV units decreased. In particular, when C/N was >12, the mole fraction of HV units decreased linearly as C/N increased. When VA was utilized as the sole carbon source and C/N was fixed at 4, P(HB-co-HV) with the highest mole fraction of HV units (67 mol%) was achieved. From these results, it was shown that both C/N and the mole ratio of BA to VA in the feed medium should be well controlled for an optimal production of P(HB-co-HV) with the desired value of the mole fraction of HV units. When the addition ratio of butyric acid was 50 wt% of total fatty acids, a maximum production strategy for P(HB-co-HV) was developed and realized experimentally, which was based on a model of the relationship between mu and rho.     

MS fragment isotope ratio analysis for evaluation of citrus essential oils by HRGC-MS

To evaluate the origin of citrus essential oils, the isotope ratio of fragment peaks on HRGC-MS of the volatile compounds from various citrus oils was measured. The MS fragment ratio was found by the ratio of fragment peak intensity, m+1/m (m/z). This ratio reflects the isotope effect of volatile compounds, that is, it provides information about locality, quality, and species for essential oils. Multivariate analysis based on the MS fragment ratio of monoterpene hydrocarbons clearly distinguished three citrus species, yuzu, lemon, and lime. The carbonyl fractions were also extracted from citrus essential oils by the sodium hydrogensulfite method. The isotope ratio of MS fragments of octanal, nonanal, and decanal was also examined. The results suggest that there was no significant difference in the individual fragment isotope ratios of the three aldehydes.     

Characterization and crystal structure of cadmium(II) halide complexes with amino acids and their derivatives VI. The comparison of crystal structures of cadmium(II) halide complexes with three kinds of piperidine carboxylic acids

Six cadmium(II) halide complexes with dl-piperidine-2-carboxylic acid (DL-Hpipe-2), dl-piperidine-3-carboxylic acid (DL-Hpipe-3), and piperidine-4-carboxylic acid (Hpipe-4), have been prepared and characterized by means of IR and Raman spectra and thermal analysis. The crystal structures of [CdCl2(DL-Hpipe-2)(H2O)], [CdBr2(DL-Hpipe-3)], and [CdCl2(Hpipe-4)] have been determined by X-ray diffraction. These three complexes have one-dimensional polymer structures bridged by halide atoms. The crystal of [CdCl2(DL-Hpipe-2)(H2O)] is orthorhombic with the space group Pca2(1). The cadmium atom is in an octahedral geometry, ligated by a carboxyl oxygen atom, two bridging chlorine atoms, a terminal chlorine atom, a water molecule and a carboxyl oxygen atom of a neighboring molecule. The carboxyl oxygen atoms of DL-Hpipe-2 are coordinated to two cadmium atoms. The unit cell consists of two types of one-dimensional polymer structures: [CdCl2(D-Hpipe-2)(H2O)] and [CdCl2(L-Hpipe-2)(H2O)]. Therefore, it is better to write [CdCl2(DL-Hpipe-2)(H2O)] as [CdCl2(D-Hpipe-2)(H2O)][CdCl2(L-Hpipe-2)(H2O)]. The crystal structure of [CdBr2(DL-Hpipe-3)] is monoclinic with space group P2(1). The cadmium atom is in a distorted octahedral geometry ligated by two carboxyl oxygen atoms and four bridging bromine atoms. This complex consists of either D-Hpipe-3 or L-Hpipe-3. Therefore [CdBr2(DL-Hpipe-3)] is written as [CdBr2(D or L-Hpipe-3)]. The crystal of [CdCl2(Hpipe-4)] is monoclinic with space group P2(1)/n. The structure is similar to that of [CdBr2(D or L-Hpipe-3)].     

Isolation and characterization of four VIP-related peptides from red-bellied newt, Cynops pyrrhogaster

Four novel bioactive peptides were isolated from the red-bellied newt, Cynops pyrrhogaster, using a bioassay system monitoring the rectum contraction of the Japanese quail, Coturnix japonica. As these peptides are structurally related to vasoactive intestinal polypeptide (VIP), we termed these peptides newt VIP-related peptides 1, 2, 3, and 4 (NVRP-1, -2, -3, and -4). The primary sequences of these peptides were determined to be HSDAVFTDNYSRLLGKTALKNYLDGALKKE (NVRP-1), HSDAVFTDNYSRLLAKTALKNYLDGALKKE (NVRP-2), HSDAVFT-DNYSRLLGKIALKNYLDEALKKE (NVRP-3), and HSDAVFTDNYSRLLGKT-ALKNYLDSALKKE (NVRP-4). The N-terminal regions of these NVRPs possessed homology at the amino-acid level to various VIP, while the NVRP C-termini differed from VIPs significantly. All of the VIP consist of 28 amino-acid residues with amidated forms at the C-termini, whereas NVRPs possess 30 amino-acid residues and have free forms at the C-termini. NVRPs exert relaxant activities on isolated quail rectums in a dose-dependent manner, with threshold concentrations between 1 x 10(-8) and 3 x 10(-8) M. NVRPs also exhibited potent relaxant activities acting on the newt duodenum at 3 x 10(-8) M. As yet, this is the first isolation of biologically active VIP-related peptides from urodele.     

Vitamin A prevents the irreversible proliferation of vaginal epithelium induced by neonatal injection of keratinocyte growth factor in mice

Exposure of female mice to estrogen during the perinatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. However, the occurrence of such irreversible vaginal changes is blocked by concurrent vitamin A treatment. Neonatal exposure to keratinocyte growth factor (KGF), which is a paracrine mediator of epithelial-mesenchymal interactions, also induces the persistent proliferation and cornification of the vaginal epithelium in adult mice. This study was designed to examine whether concurrent administration of vitamin A inhibits the development of the irreversible vaginal changes in mice exposed neonatally to KGF. The vaginal epithelium in ovariectomized 35-day-old mice given 5 microg of KGF for 3 days after birth possessed a significantly larger number of layers and increased thickness as compared to that in control mice. Concurrent injections of 100 IU of vitamin A acetate inhibited the occurrence of the irreversible proliferation of the vaginal epithelium. These changes were equal to the results observed when 20 micro g of estrogen with or without vitamin A acetate was administered for 5 days after birth. Unlike the case of estrogen treatment, the effect of neonatal treatment with KGF seemed to appear after a latent period, since the vaginal epithelium did not show proliferation soon after the treatment. We discuss the inhibitory effect of VA on the irreversible vaginal changes induced by neonatal KGF treatment with reference to endocrine disruption by neonatal estrogen exposure.     

Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Effect of molecular structure on thermodynamic properties of carbohydrates. A calorimetric study of aqueous di- and oligosaccharides at subzero temperatures

For aqueous solutions of di- and oligosaccharides thermodynamic properties have been investigated at subzero temperatures using differential scanning calorimetry. The amount of unfrozen water observed is found to increase linearly with the glass transition temperatures of anhydrous carbohydrates. Furthermore, the amount of unfrozen water shows a linear relationship with known solution properties of aqueous carbohydrates, such as partial molar compressibility and heat of solution. The different effectiveness among various di- and oligosaccharides to avoid ice formation is associated with the combination of constitutive monosaccharides and attendant molecular structure features including the position and type of the glycosidic linkage between the constituent units. More unfrozen water is induced in the presence of a carbohydrate having a poorer compatibility with the three-dimensional hydrogen-bond network of water. A series of these results obtained imply that there is a common key of carbohydrate stereochemistry governing several different thermodynamic amounts of a given system involving carbohydrates. In this context, a modified stereospecific-hydration model can be used to interpret the present results in terms of stereochemical effects of carbohydrates.