Search PubMed⌕ Search

Biomedical subjects

H Nagase

Publications and source records attributed to H Nagase.

At least 217 records · Page 12Linked to original sources
PubMed1996

DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats.

We characterized the antithrombotic, haemorrhagic, and ex vivo anticoagulant effects of a recently identified depolymerized holothurian glycosaminoglycan (DHG), and compared these effects with those of unfractionated heparin (UFH), low molecular weight heparin (LMWH), and dermatan sulfate (DS). In thrombin-induced venous thrombus formation in rats, DHG had a significant preventive effect at 0.3 mg/kg or more at 5 min after i.v., administration. UFH, LMWH, and DS also showed a significant antithrombotic effect at 0.3, 0.3, and 1 mg/kg, respectively, under the same experimental conditions. After rat tail transection, DHG, UFH, LMWH, and DS prolonged the bleeding time significantly at 10, 1, 1, and 10 mg/kg, respectively, at 5 min after i.v., injection. Therefore, DHG exerts its antithrombotic effect with less bleeding than UFH and LMWH in experimental animals. DHG prolonged the activated partial thromboplastin time in a dose-dependent manner at 0.3-3 mg/kg, at which dose an antithrombotic effect was exhibited without any significant haemorrhagic effect. All of these glycosaminoglycans prolonged thrombin clotting time markedly at their haemorrhagic doses.

Animals↗
PubMed1996

Degradation of cross-linked fibrin by matrix metalloproteinase 3 (stromelysin 1): hydrolysis of the gamma Gly 404-Ala 405 peptide bond.

Matrix metalloproteinases (MMPs) can degrade a number of proteins that constitute the extracellular matrix. Previous studies have shown that atherosclerotic plaques contain substantial amounts of fibrin(ogen)-related antigen, and more recently, MMPs have been identified in such lesions. The hypothesis that MMPs play a role in the degradation of fibrinogen (Fg) and cross-linked fibrin (XL-Fb) was investigated. Fibrinogen became thrombin-unclottable when treated with matrix metalloproteinase 3 (MMP-3, stromelysin 1) but not with matrix metalloproteinase 2 (MMP-2, gelatinase A). Incubation of XL-Fb clots (made with 125I-Fg) with MMP-3 resulted in complete lysis after 24 h. A D monomer-like fragment was generated by MMP-3 degradation of fibrinogen, XL-Fb, and fragment DD. Immunoreactivity with monoclonal antibody (MoAb)/4-2 (anti-gamma 392-406) but not with MoAb/4A5 (anti-gamma 397-411) suggested that a major cleavage site was within the sequence participating in the cross-linking of two gamma-chains. NH2-terminal sequence analysis of they gamma-chain of the D monomer-like fragment and of a dipeptide isolated from the MMP-3 digest of XL-fibrin identified the hydrolysis of the gamma Gly 404-Ala 405 peptide bond. These data indicate that the degradation of Fg and XL-Fb by MMP-3 is specific and different from plasmin. This mechanism of fibrinolysis might be of relevance in wound healing, inflammation, atherosclerosis, and other pathophysiological processes.

Amino Acid Sequence↗
PubMed1996

Deletion of CD4+ T cells by mouse mammary tumor virus (FM) superantigen with broad specificity of T cell receptor beta-chain variable region.

We previously identified a superantigen from the exogenous mouse mammary tumor virus carried by FM mice [MMTV (FM)], which can preferentially activate V beta 8.2+ CD4+ T cells by subcutaneous injection. In the present study we investigated the effect of neonatal infection with the virus on the T cell receptor (TCR) beta-chain variable region (V beta) repertoire, T cell immune response, and development of experimental allergic encephalomyelitis (EAE). The infection, surprisingly, resulted in deletion of a large portion of CD4+ T cells including V beta 2+, 6+, 8.1+, 8.3+, and 14+ CD4+ T cells in addition to V beta 8.2+ CD4+ T cells. Nevertheless, the infection marginally affected T cell immune response to various antigens such as ovalbumin (OVA) and alloantigen except the abrogated response to anti-V beta 8.2 antibody-mediated receptor cross-linking. Moreover, the infection exerted a protective effect on the development of EAE in (PL/J x SJL)F1 mice. Thus, MMTV (FM) superantigen has the ability to delete a large portion of CD4+ T cells with broad TCR V beta specificity, including V beta 8.2+ CD4+ T cells, and may have potential as a therapeutic agent against autoimmune diseases.

Animals↗
PubMed1996

Agonist and antagonist properties of dihydroetorphine for mu-opioid receptors in mice.

To test the hypothesis that dihydroetorphine has opioid antagonist properties, we examined the effects of dihydroetorphine on the antinociceptive effect of morphine. The antinociceptive response was evaluated by recording the latency in the tail-flick test using radiant heat as a stimulus. The antinociceptive effect of dihydroetorphine reached its peak 30 min after administration, and remained significant up to 90 min after administration. On the other hand, when dihydroetorphine was injected i.c.v., the antinociceptive effect of dihydroetorphine reached its peak 15 min after administration, and a significant antinociceptive effect disappeared within 90 min after administration. When morphine was administered 30 min after dihydroetorphine, the antinociceptive effect of morphine was significantly enhanced. In contrast, the antinociceptive effect of morphine was significantly reduced when morphine was administered 120 min after either i.p. or i.c.v. administration of dihydroetorphine. This antagonistic effect remained significant up to 6 h after administration of dihydroetorphine, and then gradually decreased. However, dihydroetorphine had no significant effect on the antinociceptive effect of either trans-(+/-)-3,4-dichloro-N-methyl-N-(2(1-pyrrolidinyl) cyclohexyl)-benzeneacetamide (U-50, 488H), a kappa-opioid receptor agonist, or [D-Pen2.5]enkephalin (DPDPE), a delta-opioid receptor agonist. These results suggest that dihydroetorphine may have a reversible antagonist effect for mu-opioid receptors, but not for kappa- or delta-opioid receptors, when its agonistic activity disappears.

Analgesics, Opioid↗
PubMed1996

Fluorescence quenching studies of matrix metalloproteinases (MMPs): evidence for structural rearrangement of the proMMP-2/TIMP-2 complex upon mercurial activation.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which are secreted from cells as zymogens and can be activated by treatment with organomercurial reagents or limited proteolysis. The proenzyme forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are found in complex with tissue inhibitor of metalloproteinases (designated proMMP-2/ TIMP-2 and proMMP-9/TIMP-1, respectively). The proposed mechanism of activation by mercurial compounds involves the induction of a conformational change in the zymogen which leads to propeptide autoprocessing. To investigate the possibility of conformational differences in MMPs, solute quenching of MMP intrinsic fluorescence was used to probe the relative exposure of tryptophan residues in latent and mercurial-activated MMPs. Our data demonstrate that fluorescence quenching of the proMMP-2/TIMP-2 complex by either acrylamide or iodide is significantly increased following mercurial activation. In contrast, no significant change in tryptophan accessibility accompanies mercurial treatment of either proMMP-2 or TIMP-2 alone, or mercurial-activated MMP-2 mixed with TIMP-2. To determine whether the enhanced fluorescence quenching was unique to the activated proMMP-2/TIMP-2 complex, similar experiments were performed using MMP-1, MMP-3, and MMP-9/TIMP-1 complex. In all cases, both latent and mercurialtreated MMPs exhibited similar fluorescence quenching profiles, suggesting that there are no significant conformational differences between the zymogen and activated forms of MMP-1, -2, -3, or -9/TIMP-1. The enhanced fluorescence quenching observed with mercurial-treated proMMP-2/TIMP-2 is indicative of increased exposure of a previously buried tryptophan residue(s), providing evidence for a structural rearrangement of the activated complex. These data, together with our previous biochemical observation that mercurial treatment of proMMP-2/TIMP-2 exposes the MMP-2 active site without propeptide processing (Y. Itoh et al. (1995) Biochem. J. 308, 645-651), suggest that the activated proMMP-2 in the complex may represent a transitional conformational intermediate in MMP activation.

Acrylamide↗
PubMed1996

Tumor necrosis factor alpha (TNFalpha) induces pro-matrix metalloproteinase 9 production in human uterine cervical fibroblasts but interleukin 1alpha antagonizes the inductive effect of TNFalpha.

We have examined the regulation of precursor of matrix metalloproteinase 9 (proMMP-9)/progelatinase B production by tumor necrosis factor alpha (TNFalpha) and interleukin 1alpha (IL-1alpha) using human uterine cervical fibroblasts. TNFalpha, but not IL-1alpha, induces the production of proMMP-9 in the cervical cells. IL-alpha, however, suppresses the TNFalpha-induced proMMP-9 production. 12-0-tetradecanoylphorbol 13-acetate (TPA) also stimulates the cervical cells to produce proMMP-9, and IL-1alpha synergistically enhances its production. TNFalpha-induced proMMP-9 production is not mediated by protein kinase C (PKC), whereas the effect of IL-1alpha is through PKC. By contrast, proMMP-3/prostromelysin 1 is up-regulated by TNFalpha or TPA in the presence of IL-1alpha, whose modulation is PKC-dependent. The suppressive effect of IL-1alpha on the TNFalpha-induced proMMP-9 production is a new biological effect of IL-1 on MMP production.

Alkaloids↗
PubMed1996

Relaxed specificity of matrix metalloproteinases (MMPS) and TIMP insensitivity of tumor necrosis factor-alpha (TNF-alpha) production suggest the major TNF-alpha converting enzyme is not an MMP.

Tumor necrosis factor-alpha is released from cells by a proteolytic cleavage. Previous work suggested that a specific, non-matrix metalloproteinase carries out this cleavage, but matrix metalloproteinases have also been implicated. In this paper, we report that none of the matrix metalloproteinases tested cleaved peptide substrates as specifically as the non-matrix metalloproteinase. A matrix metalloproteinase did process tumor necrosis factor-alpha extracted from COS cells, but neither tissue inhibitor of metalloproteinases-1 nor -2 blocked tumor necrosis factor-alpha processing by human monocytes. Moreover, tissue inhibitor of metalloproteinases-1 had at most a partial effect on the in vivo release of the cytokine in mice. We conclude that a non-matrix metalloproteinase is the major physiological tumor necrosis factor-alpha convertase.

ADAM Proteins↗
PubMed1996

Effect of metallothioneins on transformation of gelatinase A from human fibroblast WI-38 cells.

The effect of metallothioneins (MTs) on gelatinase A activity was investigated. The collagenolytic activity of gelatinase A from human fibroblast WI-38 cells was enhanced by the addition of MTs. This enhancement may be caused by the transformation of the inactive 62 kDa intermediate species of gelatinase A to the 59 kDa active enzyme. This enhancement was also observed in the conditioned medium of WI-38 cells exposed to heavy metals, but intracellular 72 kDa pro-gelatinase A did not change. Furthermore, degradation of gelatinase A occurred in the reaction between gelatinase A with substrate and MTs. Our results suggest that MTs may be an endogenous activator of gelatinase A, and may provide a host factor in cancer metastasis.

Animals↗
PubMed1996

Interaction of a new depolymerized holothurian glycosaminoglycan with proteins in human plasma.

In a rat template bleeding model, depolymerized holothurian glycosaminoglycan (DHG) prolonged bleeding time at 30 mg/kg i.v. but unfractionated heparin (UFH) had the same effect at 1 mg/kg i.v., indicating that DHG is much less bleeding than UFH. To characterize this difference, we examined the affinity of DHG for plasma proteins by means of a glycosaminoglycan-conjugated cellulofine column in comparison with that of UFH. The DHG column strongly bound factor V, factor IX, protein S, histidine-rich glycoprotein, platelet factor 4 (PF4), beta-thromboglobulin, von Willebrand factor, fibronectin, and heparin cofactor II, but did not bind fibrinogen, prothrombin, factor VII, protein C, antithrombin III (ATIII), plasminogen or alpha 2-plasmin inhibitor. The profile of protein binding to the UFH column was almost the same as that of the DHG column except that ATIII showed affinity for UFH. One of the reasons why DHG caused much less bleeding than UFH is thus suggested to be the differences in their affinity for ATIII in plasma.

Animals↗
PubMed1996

Degradation of interleukin 1beta by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1beta, but not IL-1alpha, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1beta was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1beta by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1beta stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1beta.

Amino Acid Sequence↗
PubMed1996

Involvement of the opioid system in the anxiolytic effect of diazepam in mice.

In the present study, the anticonflict effect of diazepam was significantly abolished by pretreatment with naloxone, beta-funaltrexamine or nor-binaltorphimine but not naltrindole, using a Vogel-type conflict paradigm in mice. However, naloxone alone had a significant proconflict effect, and beta-funaltrexamine alone tended to produce a proconflict effect. Spontaneous drinking behavior was not affected by treatment with diazepam and nor-binaltorphimine. In addition, nor-binaltorphimine had no effect on diazepam-induced motor incoordination, hypothermia or anticonvulsant action, respectively. Moreover, the stable dynorphin analog E2078 ([N-methyl-Tyr1, N-alpha-methyl-Arg7-D-Leu8]dynorphin A-(1-8) ethylamide) and the highly selective kappa-opioid receptor agonist U50,488H (trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenacetamide++ + methanesulfonate hydrochloride) produced a significant anticonflict effect, which was completely antagonized by pretreatment with nor-binaltorphimine. These findings suggested that the kappa-opioid system may play an important role in the anxiolytic effect of benzodiazepine and the regulation of anxiety.

Analgesics, Non-Narcotic↗
PubMed1996

Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83.

The precursor of matrix metalloproteinase 3 (MMP-3/ stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His82-Phe83 bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His82 or Phe83 to Arg to obtain a stable MMP-3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha 2-macroglobulin. However, the SH group of Cys75 of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys75. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr85, whose activity and substrate specificity were similar to those of the 46-kDa MMp-3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3.

Amino Acid Sequence↗
PubMed1996

Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.

Methods are described for producing an active amino-terminal domain of tissue inhibitor of metalloproteinases-1 (N-TIMP-1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis. The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2 and 3 is 6-8-fold less than that of the recombinant full-length protein, indicating that deletion of the C-terminal domain reduces the free energy of interaction by < 10%.

Amino Acid Sequence↗
PubMed1996

Antinociceptive and antitussive effects of morphine in the DA-bg/bg (beige) rat.

Antinociceptive and antitussive effects of morphine were studied in DA-bg/bg (Beige) rats. Intraperitoneal administration of morphine (10 mg/kg) produced a marked antinociceptive effect, in the tail-flick test, in Beige rats and DA rats, a progenitor strain rats. There was no significant difference in the peak antinociceptive effect of morphine between Beige rats and DA rats. The antinociceptive effect of morphine in both Beige rats and DA rats was significantly reduced following pretreatment with a low dose (0.3 mg/kg, i.p.) of naloxone or naltrexonazine (1 mg/kg, s.c.), a selective mu 1-opioid receptor antagonist. Morphine suppressed coughs dose dependently at doses between 0.3-3 mg/kg, i.p., in Beige rats and between 0.1-1.0 mg/kg, i.p., in DA rats. The antitussive potency of morphine in Beige rats was less than that in DA rats. The antitussive effect of morphine was significantly antagonized by pretreatment with naloxone (0.3 mg/kg, i.p.) in both Beige rats and DA rats. However, pretreatment with naltrexonazine (1 mg/kg, s.c.), a selective mu 1-opioid receptor antagonist, had no effect on the antitussive effect of morphine. These results suggest that Beige rats are hyporesponsive to the mu 2-opioid receptor-mediated antitussive effect, but not to the mu 1-opioid receptor-mediated antinociceptive effect.

Analgesics, Opioid↗
PubMed1996

Translational augmentation of pro-matrix metalloproteinase 3 (prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs induced by epidermal growth factor in human uterine cervical fibroblasts.

The mechanisms by which epidermal growth factor (EGF) enhances the production of pro-matrix metalloproteinase 3 (proMMP-3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 were investigated using human uterine cervical fibroblasts. The treatment of the cells with EGF for 24 h resulted in about 5-6-fold increase in the production of proMMP-3 and TIMP-1 compared with the untreated control cells. This increase was accompanied by an increase of proMMP-3 and TIMP-1 mRNAs. However, an about 3- and 2-fold increase in the production of proMMP-3 and TIMP-1, respectively, was observed as early as 1 h after the treatment of the cells with EGF, and it was not accompanied by any apparent increase in proMMP-3 and TIMP-1 mRNAs. This early effect of EGF on the enhanced production of proMMP-3 and TIMP-1 was not inhibited by actinomycin D, even though actinomycin D inhibited the synthesis of the total RNA in both the EGF-treated and untreated cells. These results indicate that EGF enhances the apparent production of proMMP-3 and TIMP-1 by two mechanisms: one by the accelerated translation of their mRNAs; and the other by the enhanced transcription of their genes. The former event takes place much earlier than the latter.

Cells, Cultured↗
PubMed1996

Effect of galanin and enterostatin on sympathetic nerve activity to interscapular brown adipose tissue.

The effects of galanin and enterostatin on sympathetic activity have been examined in rats using electrophysiological techniques. Galanin, in doses of 25-300 pmol, and enterostatin, in doses of 0.5-10 nmol, were injected into the third ventricle of anesthetized male Sprague-Dawley rats in 1-microliter volumes. Galanin produced a dose-dependent suppression (ranging between 20 and 80%) of sympathetic firing rate of nerves innervating interscapular brown adipose tissue. In rats fed a chow diet, injection of enterostatin produced only a transient 10% rise in firing rate which returned to baseline within 10-15 min. In contrast, animals fed a high-fat diet showed a dose-dependent increase in firing rate lasting for 60 min. The results of this experiment are consistent with the hypothesis that food intake and sympathetic nervous system activity have a reciprocal relationship. The implications of this relationship are discussed.

Adipose Tissue, Brown↗
PubMed1996

Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides.

The matrix metalloproteinase (MMP)/matrixin family has been implicated in both normal tissue remodeling and a variety of diseases associated with abnormal turnover of extracellular matrix components. To better understand MMP behaviors and to aid in the design of MMP inhibitors, a variety of sequence specificity studies have been performed using collagen sequence-based peptides and MMP family members. Results of these studies have been valuable for defining the differences in MMPs and for creating fluorogenic substrates that can continuously monitor MMP activity. However, these studies have also demonstrated that these peptides may not be very good models of native MMP substrates, and that the additivity principle is not always applicable for designing synthetic MMP substrates.

Amino Acid Sequence↗