PubMed Health⌕ Search

Biomedical subjects

K Nokihara

Publications and source records attributed to K Nokihara.

At least 19 recordsLinked to original sources
PubMed2001

Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates.

In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.

Aged↗
PubMed2001

High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method.

A high-throughput method for determination of total nitrogen content has been developed. The method involves decomposition of samples, followed by trapping and quantitative colorimetric determination of the resulting ammonia. The present method is rapid, facile, and economical. Thus, it can replace the classic Kjeldahl method through its higher efficiency for determining multiple samples. Compared to the classic method, the present method is economical and environmentally friendly. Based on the present method, a novel reactor was constructed to realize routine high-throughput analyses of multiple samples such as those found for pharmaceutical materials, foods, and/or excrements.

Ammonia↗
PubMed1998

Quantitative determination of biological sulfhydryl groups by postcolumn derivatization and elucidation of microheterogeneity of serum albumins.

A quantitative analytical system for biological sulfhydryl compounds has been developed using an ion-pair reagent with isocratic elution and an on-line postcolumn derivatization with Ellman-type reagents. As human or bovine serum albumin has 35 cysteinyl residues, one cysteinyl residue exists as a free sulfhydryl moiety, and this gives rise to the microheterogeneity in serum albumin. Here we report for the first time the quantitative characterization of the microheterogeneity of serum albumin. Cysteine was found to be the major molecule attached to the sulfydryl group of the serum albumins. Although glutathione could not be detected, the Cys-Gly element of glutathione was found. Freshly prepared human serum albumin from healthy volunteers contained 0.46 nmol of Cys/mL of serum, 0.24 nmol of Cys-Gly/mL of serum, and very small amounts of glutathione (0.02 nmol/mL).

Adult↗
PubMed1998

Antioxidative Properties of Histidine-Containing Peptides Designed from Peptide Fragments Found in the Digests of a Soybean Protein.

The properties of 22 synthetic peptides containing histidine, which were designed on the basis of the antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, were examined with regard to their antioxidative activity against the peroxidation of linoleic acid and the scavenging effects on active oxygen and free radical species. The antioxidative activities of these peptides in an emulsion oxidation system using 2,2'-azobis(2-amidinopropane) dihydrochloride as a radical initiator correlated well within an aqueous system. Although the histidine-containing peptides had a quenching activity on singlet oxygen, they did not show antioxidative activity in an 2,2'-azobis(2,4-dimethylvaleronitrile)-induced oxidation system or scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and superoxide. The metal-ion chelating activities and the hydrophobicities of these peptides showed no direct correlation with their antioxidative activities. Leu-Leu-Pro-His-His was modified with a hydroxyl radical in an aqueous ethanol system during the peroxidation of linoleic acid.

Journal Article↗
PubMed1998

Crucial role of N-terminal residue of binding peptides in recognition of the monoclonal antibody specific for the peptide-HLA-B5, -B35 complex.

The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B*3501 and HLA-B*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B*3501 and -B*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex.

Amino Acid Sequence↗
PubMed1998

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference.

HLA-B*3501 and -B*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B*3501 and HLA-B*5101 molecules, we generated HLA-B*3501 mutant molecules carrying Tyr at residue 116 (B*3501-116Y) and tested the binding of a panel of nonamer peptides to the B*3501-116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B*3501-116Y molecules was much higher than that to HLA-B*3501 and HLA-B*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B*3501 and HLA-B*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B*3501 and HLA-B*5101 may be responsible for the low peptide binding property of the latter.

Binding Sites↗
PubMed1998

Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle.

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.

Amino Acid Sequence↗
PubMed1998

Coexistence of proguanylin (1-15) and somatostatin in the gastrointestinal tract.

In order to identify proguanylin-secreting cells, we have raised an antiserum against the synthetic fragment of human proguanylin (1-15) and have examined the proguanylin-positive cells in the human and rat gastrointestinal tract by immunohistochemical methods. Numerous proguanylin (1-15)-immunoreactive cells were found in the gastrointestinal tract. They were either pyramidal or spindle shaped in the stomach. Spindle-shaped cells, frequently possessing long slender processes, were located at the base of the pyloric epithelium and did not extend to the lumen. In the duodenum and jejunum, these cells were mostly pyramidal in shape and often had a slender process towards the lumen. The immunostaining was completely blocked by the human proguanylin (1-15) fragment. Paneth and goblet cells were negative against this antiserum. The number of serotonin-positive cells was much larger than that of proguanylin-positive cells in all the segments tested. The number of proguanylin-positive cells decreased from the jejunum to the ileum and very few cells were observed in the colon. In contrast to serotonin-positive cells, most somatostatin-positive cells were also positive for proguanylin. Thus, proguanylin (1-15) or its related protein appears to coexist with somatostatin in intestinal endocrine D cells which may be a source of circulating proguanylin. Proguanylin, like somatostatin, may also regulate intestinal function as a local regulator.

Animals↗
PubMed1997

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer.

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4-12 and 7-15, and its disulfide isomer, with disulfides positions 4-15 and 7-12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by 1H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.

Amino Acid Sequence↗
PubMed1997

Complete determination of disulfide forms of purified recombinant human serum albumin, secreted by the yeast Pichia pastoris.

In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.

Amino Acid Sequence↗
PubMed1997

Effect and distribution of intravenously injected 125I-guanylin in rat kidney examined by high-resolution light microscopic radioautography.

125I-guanylin was injected intravenously into rats, and their kidney and intestinal tract were processed for light microscopic radioautography using semithin sections to examine the binding sites. Various doses of unlabeled guanylin were also injected to examine the morphological effects of guanylin on the kidney. Dense labeling of silver grains due to 125I-guanylin were observed only in the kidney. In the cortex, silver grains were localized on the luminal side of the proximal tubules at 5-30 min. In the medulla, silver grains appeared at the basal side of the collecting ducts, capillaries and loops of Henle after 5 min. Silver grains then accumulated in the cytoplasm of the collecting ducts after 10 min, and disappeared after 30 min. The cell height of the inner medullary collecting ducts (IMCD) decreased and their luminal spaces increased dose-dependently 5 min after the injection of both labeled and unlabeled guanylin. These structural changes returned to control levels within 30 min. These results indicate a high density localization of guanylin receptors on the luminal surface of proximal tubules in the renal cortex and also rapid excretion of guanylin through the IMCD. The morphological changes of the IMCD suggest a diuretic effect of guanylin.

Animals↗
PubMed1996

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

Animals↗
PubMed1996

NMR spectroscopic evidence that helodermin, unlike other members of the secretin/VIP family of peptides, is substantially structured in water.

The structure in water and additionally in 50% trifluoroethanol (TFE) solution of helodermin, an amidated peptide consisting of 35 amino acids, was elucidated by 2D 1H NMR spectroscopy initially from H alpha chemical shifts and qualitative NOE data. Detailed structures were calculated from the quantitative NOE data which were used as distance restraints in molecular dynamics and energy minimization calculations. Regions of stable secondary structure were defined from the resulting final peptide conformations using a new fitting program that takes into account the summed RMS differences between all structures for short segments of 2-5 residues in length. This procedure allows a reasonably objective method of defining the edges of stable structure. In contrast to other members of the secretin/VIP family of peptides, helodermin shows a defined secondary structure in water alone and possesses an alpha-helix from Glu-9 to Leu-23 that was further stabilized and slightly extended (Phe-6 to Ala-24) on addition of TFE. The N- and C-termini were unstructured in both solutions. Such features, in particular the observation of a linear helix 18 +/- 2 residues in length, are common to other members of the family and become more pronounced in hydrophobic environments. The data provide further circumstantial evidence that an alpha-helix conformation is necessary for receptor binding. The prolonged physiological action of helodermin, compared to its C-terminal deletion analogues and VIP, is at least in part due to the unusual stable secondary structure.

Amino Acid Sequence↗
PubMed1996

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding.

HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 - 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.

Amino Acid Sequence↗
PubMed1996

Distribution of immunoreactive atrial and brain natriuretic peptides in the heart of the chicken, quail, snake and frog.

The distribution of atrial natriuretic peptide (ANP)- and brain natriuretic peptide (BNP)-granules was examined immunohistochemically and ultrastructurally in the hearts of the chicken, Japanese quail, Japanese rat snake and bull-frog. Moreover, natriuretic peptide (NP)-granules in the cardiocytes were analyzed by ultrastructural morphometry. Immunohistochemically, ANP-immunoreactivity (IR) was not detected in any cardiocytes, but BNP-IR was detectable in most atrial and ventricular cardiocytes of both chicken and quail. In the snake, ANP-IR was seen in most atrial and ventricular cardiocytes, which showed traces and negative in BNP-IR, respectively. Both ANP- and BNP-IR were detected in the atrial and ventricular cardiocytes in the frog. Ultrastructurally, most of NP-granules were found in the perinuclear region in the chicken, quail and snake atrium, but the frog atrial cardiocytes had granules generally dispersing widely in the cell. By ultrastructural morphometry, the number of granules in the atrial cardiocyte was greatest in the frog, followed by the snake, and chicken or quail, in this order. The diameter of granules in the atrial cardiocyte was largest in the snake and reduced via the frog to the chicken or quail. In the ventricular cardiocytes of all species, the number and size of granules were significantly less than that in the atrial ones. These results indicated that the hearts of the chicken and quail contain only BNP, and that there are two different natriuretic peptides, ANP and BNP, in the snake and frog hearts.

Animals↗