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K Titani

Publications and source records attributed to K Titani.

At least 55 records · Page 3Linked to original sources
PubMed1996

Primary structure of alboaggregin-B purified from the venom of Trimeresurus albolabris.

The complete amino acid sequences of alpha and beta subunits of alboaggregin-beta are presented. The alpha and beta subunits were separated by reversed-phase HPLC after reduction and S-pyridylethylation, and their sequences were determined by analysis of peptides generated by enzymatic or chemical digestion. The alpha and beta subunits consist of 133 and 123 amino acid residues, respectively. The sequences are highly homologous to each other (41.4% identity) and also to those of the alpha and beta subunits of botrocetin (a von Willebrand factor modulator) and the A and B chains of factor IX/X binding protein from other snake venoms. It is also homologous to C-type lectins with a homodimeric structure, but it shows no lectin-like activity.

Amino Acid Sequence↗
PubMed1996

A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.

The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.

Amino Acid Sequence↗
PubMed1995

The signal modulator protein 14-3-3 is a target of sphingosine- or N,N-dimethylsphingosine-dependent kinase in 3T3(A31) cells.

Phosphorylation of a 28 kDa protein in 3T3(A31) cells upon addition of semi-purified kinase was induced only by sphingosine (Sph) or N,N-dimethylsphingosine (DMS), but not by 17 other lipids tested. The 28 kDa protein, a major target of Sph- or DMS-dependent kinase, is now identified as 14-3-3 protein, which modulates a number of key protein kinases involved in regulation of intracellular signaling (Aitken A, Trends Biochem Sci 20:95-97, 1995; Morrison D, Science 266:56-57, 1994). Thus, Sph and DMS modulate the function of a versatile modulator controlling a number of signaling molecules.

14-3-3 Proteins↗
PubMed1995

The complete amino acid sequence of a mannose-binding lectin from "Kidachi Aloe" (Aloe arborescens Miller var. natalensis Berger).

The complete amino acid sequence of a mannose-binding lectin purified from the leaf skin of "Kidachi Aloe" (Aloe arborescens Miller var. natalensis Berger) is presented. The 109-residue sequence of the subunit was determined by analysis of peptides of the intact or S-pyridylethylated protein generated by digestion with cyanogen bromide, BNPS-skatole, Achromobacter protease I, or trypsin. The subunit contains an intrachain disulfide bridge. The sequence is highly homologous to that of a mannose-binding lectin from snowdrop bulb.

Aloe↗
PubMed1995

Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation.

A new platelet antagonist, tokaracetin, was isolated from the venom of Trimeresurus tokarensis by ion-exchange chromatography, heparin-Sepharose chromatography and hydrophobic HPLC. The purified protein showed an apparent molecular mass on SDS/PAGE of 28.9 kDa under non-reducing conditions. On reduction, 16.1 and 15.4 kDa subunits were observed, suggesting that the molecule is a heterodimer. Tokaracetin inhibited the binding of 125I-labelled bovine von Willebrand factor (vWF) and 125I-labelled human vWF in the presence of botrocetin to fixed human platelets. It did not block ADP-, collagen- or thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma (PRP), or induce platelet agglutination in PRP. On reduction, tokaracetin lost its inhibitory activity on the agglutination of fixed human platelets by bovine vWF. 125I-Tokaracetin specifically bound to washed human platelets with high affinity (Kd 3.9 +/- 1.4 nM) at 47,440 +/- 2780 binding sites per platelet. Binding of tokaracetin to fixed human platelets was reversible, and was inhibited by monoclonal antibody GUR83-35, which is directed against the N-terminal vWF-binding domain of human glycoprotein Ib (GPIb). Tokaracetin completely inhibited vWF-dependent shear-induced platelet aggregation in PRP at 3 micrograms/ml. The N-terminal amino acid sequences of tokaracetin subunits showed a high degree of identity with those of alboaggregin-B. These results suggest that this new platelet antagonist may be a useful tool in the development of specific inhibitors of the vWF-GPIb interaction.

Amino Acid Sequence↗
PubMed1995

Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress.

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.

Adenosine Triphosphate↗
PubMed1995

Amino acid sequence of a modified beta 2-microglobulin in renal failure patient urine and long-term dialysis patient blood.

We isolated and analyzed beta 2-microglobulin (BM) in urine of two renal failure patients and in ultrafiltrate from long-term dialysis patients. In both groups of patients, we found the native BM with pI 5.7 and some modified BM with pI 5.3. The amino acid sequence of the modified BM was determined and compared with that of the native BM; the N-terminal isoleucine residue was missing and the second glutamine residue was cyclized into pyroglutamic acid in the modified BM.

Amino Acid Sequence↗
PubMed1995

Identification of two 17-kDa rat parotid gland phosphoproteins, subjects for dephosphorylation upon beta-adrenergic stimulation, as destrin- and cofilin-like proteins.

We previously reported that when 32Pi-loaded rat parotid slices are incubated with the beta-adrenergic agonist isoproterenol, the level of a soluble 32P-labeled 17-kDa protein (pp17) decreases rapidly (Kanamori, T., and Hayakawa, T. (1982) Biochem. Int. 4, 517-523). Here we show that pp17 consists of two distinct phosphoproteins (pp17a and pp17b), identify their unphosphorylated forms (p17a and p17b, respectively), and provide evidence for their beta-adrenergic stimulation-induced dephosphorylation. Since p17a and p17b were predominant forms even in nonstimulated cells, peptides were generated from them with Staphylococcus aureus V8 protease or cyanogen bromide; subsequent sequencing of these peptides and homology search allowed identification of p17a and p17b as destrin- and cofilin-like proteins, respectively. Interestingly, they were also dephosphorylated in response to cholinergic stimulation. Because destrin and cofilin are actin-depolymerizing proteins whose activities are possibly regulated by their phosphorylation/dephosphorylation, the two parotid proteins reported here might be involved in cortical F-actin disruption observed in parallel with exocytotic amylase secretion.

Actin Depolymerizing Factors↗
PubMed1995

Proline-directed and non-proline-directed phosphorylation of PHF-tau.

To gain insight into the abnormal phosphorylation of PHF-tau, we have determined the phosphorylation sites by identifying phosphopeptides by means of ion spray mass spectrometry followed by sequencing of ethane-thiol-modified peptides. Nineteen sites have been identified; all but Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Eleven sites correspond to fetal type sites. Unexpectedly, 10 are non-proline-directed, whereas the others are proline-directed. Thus, the abnormal phosphorylation of PHF-tau can be considered to consist of fetal type phosphorylation and additional proline-directed and non-proline-directed phosphorylation. This non-fetal type phosphorylation may provide PHF-tau with the unusual characteristics.

Alzheimer Disease↗
PubMed1995

Developmental expression of D-galactoside-binding lectin in sea urchin (Anthocidaris crassispina) eggs.

The spatial and temporal expression of a sea urchin (Anthocidaris crassispina) egg lectin (SUEL) during early embryogenesis was studied using antiserum raised against SUEL. Western blotting analysis revealed the presence of SUEL in all stages so far examined, from unfertilized eggs to gastrula stage embryos. Immunofluorescence and immunoelectron microscopic observation showed that SUEL was stored in small electron-dense granules which migrated to the cortex within 10 min after fertilization. SUEL was localized in the cortical cytoplasm of the blastomere during cleavage stages and subsequently migrated to the outer surface of the embryo, including the invaginated portion of the gastrula. Immunoelectron microscopic study indicated that SUEL was deposited in the hyaline layer at least at the mid gastrula stage. Migration of SUEL to the cortex was significantly reduced by treatment with cytochalasin B, suggesting that actin filaments play an important role in this translocation. Exogenously added SUEL was adsorbed at the surface of unfertilized eggs and hatched embryos, but not to embryos with fertilization membrane. Lactose inhibited this adsorption, suggesting the presence of an endogenous glycoligand(s) specific for SUEL on the surface of unfertilized eggs and in the hyaline layer. We conclude that SUEL is secreted at a certain stage of embryogenesis and specifically adsorbed to the hyaline layer. Temporal changes in extraembryonic matrices caused by SUEL seem to play an important role in developmental morphogenesis.

Actin Cytoskeleton↗
PubMed1995

Immunocytochemical localization of GTP cyclohydrolase I in the brain, adrenal gland, and liver of mice.

GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, the enzymes that synthesize tyrosine, catecholamines (dopamine, noradrenaline, and adrenaline), and serotonin, respectively. We produced for the first time polyclonal antibody with highly sensitive immunoreactivity against an oligopeptide of rat enzyme, GEPERELPRPGA, by immunization of rabbits with the peptide conjugated to hemocyanin by glutaraldehyde. The specificity of the antibody was confirmed by Western blot analysis. Using this antibody specific for GCH, we observed strong GCH immunostaining in the liver cells, in the dopamine-, noradrenaline-, adrenaline-, or serotonin-containing cells of the brain, and in the adrenal gland of mice. Immunocytochemical studies revealed GCH to be localized in monoamine-containing perikarya in the periglomerular cells of the olfactory bulb, zona incerta, arcuate nucleus, ventral tegmental area, substantia nigra pars compacta, locus ceruleus, nucleus tractus solitarius, area postrema, and ventrolateral area of the medulla oblongata. GCH immunostaining was particularly strong in serotoninergic nuclei, such as dorsal and median raphe nuclei, nucleus raphe pallidus, and nucleus raphe magnus. By immunoelectron microscopy, GCH-labeled cytoplasm and microtubules in the processes were observed ultrastructurally, but no staining was found in the mitochondria, and Golgi apparatus. Immunostaining was observed neither in the group D neurons that contain only aromatic amino acid decarboxylase without tyrosine hydroxylase, nor in glial cells and endothelial cells. These results indicate the abundant presence of GCH in catecholaminergic and serotoninergic neurons as well as in the adrenal medulla and liver, where BH4 is synthesized as the cofactor of tyrosine, tryptophan, and phenylalanine hydroxylases.

Adrenal Glands↗
PubMed1995

Binding of human IgM from a rheumatoid factor to IgG of 12 animal species.

The binding of IgM from a rheumatoid factor (RF-IgM) to IgG from 12 animal species was analyzed by an ELISA system. The RF-IgM bound various animal IgG with dissimilar affinities. The binding of RF-IgM to animal IgG was inhibited by addition of protein A, which binds some animal IgG by recognizing the junctional site on CH2-CH3 domains in the Fc region. As previously reported, no significant correlation was observed between the binding of RF-IgM to IgG and the content of galactose-free oligosaccharides, which is increased in IgG of rheumatoid arthritis patients or autoimmune mice. We suggest that the crucial epitope of IgG for RF-IgM binding is not the oligosaccharide structure generated specifically in IgG of autoimmune diseases but that RF-IgM may recognize a certain protein conformation of a region in IgG near the binding site of protein A.

Animals↗
PubMed1995

Functional significance of platelet membrane glycoprotein p62 (GP VI), a putative collagen receptor.

We previously suggested that platelet 62 kDa protein (p62, GP VI) is a putative collagen receptor by the studies on platelets and antiplatelet antibody in a patient (YA) with idiopathic thrombocytopenic purpura (ITP) who showed defective collagen-induced platelet function. Herein, we confirm that p62/p57 (reduced/unreduced conditions) is a specific membrane protein recognized by YA antibody using an improved immunoprecipitation technique. To further study the functional role of this protein, a crude p57 fraction ('p57') was obtained from human platelets by serial chromatography including DEAE-Sepharose CL-6B and WGA-agarose followed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This 'p57' diminished the platelet-aggregating activity of collagen and of YA immunoglobulin G (IgG). N-terminal amino acid sequence analysis of 'p57' showed that it contained intercellular adhesion molecule 2 (ICAM-2). Immunoprecipitation experiments using platelet lysates and anti-ICAM-2 monoclonal antibody demonstrated that ICAM-2 molecules had a molecular weight similar to p62 on SDS-PAGE. However, ICAM-2 was also expressed on YA's p62-deficient platelets both on immunoprecipitation and flow cytometric analysis. Thus, ICAM-2 seems to be different from p62.

Amino Acid Sequence↗
PubMed1995

Tissue fibronectin is an endogenous ligand for galectin-1.

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.

Animals↗
PubMed1995

Fibronectin and 130-kDa molecule complex mimics snake venom botrocetin-like structure potentially modulating association between von Willebrand factor and vascular vessel wall.

We established seven hybridomas secreting monoclonal antibodies (MoAbs) against the venom from Bothrops jararaca. Six of them were demonstrated to specifically recognize botrocetin, a venom protein which binds with von Willebrand factor (vWf) and induces platelet agglutination. Two of them, BCT4-3 and BCT115-2 MoAbs, could significantly inhibit botrocetin binding with plasma vWf. BCT4-3 could react slightly with a monolayer of human endothelial cells (ECs), and BCT4-3 binding to ECs was drastically enhanced by the coexistence of human plasma in a dose-dependent manner, indicating that a biological modulator structurally resembling botrocetin is created initially on the EC surface complexed with some plasma proteins. Botrocetin-like components could be immuno-purified only by immobilized BCT4-3, but not by the other immobilized MoAbs, from umbilical vein extracts. Interestingly, the immunoisolated materials were identified to consist essentially of fibronectin (Fn) and a 130 kDa molecule, and this complex bound to vWf in the extracts. Depletion of Fn from plasma decreased BCT4-3 binding to ECs. The epitope of BCT4-3 expressed on the endothelial surface, comprising plasma Fn and the coisolated 130 kDa molecule, is proposed to be a physiological modulator structurally mimicking botrocetin, and essentially supporting vWf-binding to injured endothelium and subsequently to circulating platelets.

Antibodies, Monoclonal↗
PubMed1995

A 35 kDa mannose-binding lectin with hemagglutinating and mitogenic activities from "Kidachi Aloe" (Aloe arborescens Miller var. natalensis Berger).

A novel lectin was isolated from the leaf skin of "Kidachi Aloe" (Aloe arborescens Miller var. natalensis Berger) by sequential chromatographies on Sephadex G-25 gel filtration, DEAE ion exchange, and Superdex 75 gel filtration columns. The native lectin exhibited a molecular mass of about 35 kDa on both gel filtration on a Superdex 75 column and native-PAGE under nonreducing conditions. SDS-PAGE in the presence or absence of beta-mercaptoethanol revealed two distinct peptides with molecular masses of about 5.5 and 2.3 kDa, respectively, in addition to a major 9.2 kDa subunit, indicating the presence of a partially processed subunit. The N-terminal amino acid sequence of the intact subunit showed homology with that of snowdrop lectin. The native lectin showed hemagglutinating activity toward rabbit but not human and sheep erythrocytes, and specifically bound to mannose like snowdrop lectin did, indicating that the Aloe and snowdrop lectins are structurally and functionally similar proteins. In addition, the native lectin showed strong mitogenic activity toward mouse lymphocytes.

Aloe↗
PubMed1995

ABO blood group genotype and plasma von Willebrand factor in normal individuals.

von Willebrand factor (vWF) is a multimeric plasma protein with ABO (H) blood group sugar chains. We investigated a total of 330 plasmas from normal individuals having various ABO genotypes, with special reference to vWF antigen and its platelet glycoprotein-Ib-related biological activities, termed ristocetin cofactor (RCof) and botrocetin cofactor (BCof). RCof reflects the biological activity of higher vWF multimers, while BCof reflects that of vWF of multimers of all sizes. Plasmas from normal individuals carrying one O gene (genotypes AO and BO) had slightly, but proportionally lower levels of vWF antigen, RCof, and BCof than those carrying no O gene (genotypes AA, AB, and BB). Normal plasmas from individuals carrying two O genes (genotype OO) showed much lower values for these parameters than the other plasmas, as previously reported. However, multimeric analysis of plasma vWF antigen revealed no differences among the different genotypes.

ABO Blood-Group System↗