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Biomedical subjects

M Niwa

Publications and source records attributed to M Niwa.

At least 181 records · Page 10Linked to original sources
PubMed1997

[Changes in alpha 2-adrenoceptor binding nature in guinea-pig brain following the development of morphine dependence].

We examined the effect of morphine dependence on alpha 2-adrenoceptors in guinea-pig brain. We also studied the influence of clonidine on the naloxone-induced withdrawal signs. 1. Guinea-pigs were implanted subcutaneously with MS contin (300 mg.kg-1). Two days after implantation, the binding of 3H-UK14304 (alpha 2 selective ligand) to brain membranes prepared from morphine dependent and control animals was determined. Scatchard plot of the saturation binding data revealed an increase in Bmax values and no change in the Kd values from dependent animals. These results indicate that brain alpha 2-adrenoceptors are up-regulated in morphine dependent guinea pigs. 2. Subcutaneous injection of naloxone on the 2 nd day after implantation caused characteristic withdrawal symptoms. Clonidine has been shown to reduce some opiate withdrawal signs in morphine-dependent animals.

Adrenergic alpha-Agonists↗
PubMed1997

[Changes in opioid receptor binding nature in rat brain and spinal cord following formalin or carrageenan-induced nociception].

Previous reports demonstrated the regulation of opioid and their receptor in nociception, but it is not clear how nociceptive activation may alter opioid receptor binding nature. We determined the change of mu, delta and kappa opioid receptor types in brain and spinal cord homogenates obtained from animal models receiving nociceptive treatment. 1) Rats received a subcutaneous injection of formalin and carrageenan into planter aspect of a hindpaw. These agent-injected animals were observed for appearance of pain-related behavior (guarding of the treated paw) within 2-3 hour after treatment. Following these pain behavior, rats were decapitated and brain and spinal cord were removed rapidly. 2) The binding of 3H-DAGO (mu agonist), 3H-DPDPE (delta agonist) and 3H-EKC (kappa agonist) to brain and spinal cord membranes prepared from nociceptive treatment and control rats was determined. Using these tracer 3H-opioid ligands, we failed to see any change in the total number of opioid binding sites (Bmax values) or the affinity constant (Kd values) for binding in whole brain and spinal cord. These results indicate that in these animal models which use experimentally induced inflammation to stimulate a condition of nociception, there appears to be no alteration in the levels of mu, delta or kappa binding sites.

Animals↗
PubMed1996

Assembly of hair keratins in transfected epithelial cells.

To define the interactions required for the filament assembly of differentiation-specific keratins, active copies of mouse hair keratin mHa1 and mHb4 genes were introduced into a rat kangaroo kidney epithelial cell line (PtK2) and a rat stratified squamous epithelial cell line (rat epidermal keratinocyte). In PtK2 transient transfectants, when introduced individually or in combination, mHa1 and mHb4 formed aggregates of ring-like structures of various sizes at the perinuclear region with no evidence of organization into a keratin network. These aggregates altered the distribution of the endogenous keratins and vimentin. In most of the cells carrying the ring-like structures of mHa1 and mHb4 around the nucleus, the endogenous keratin network collapsed and localized around the nucleus. Furthermore, the densely accumulated endogenous keratin surrounded the ring-like aggregates with partial co-localization. However, when transfected into the rat epidermal keratinocytes, mHa1 and mHb4 were able to co-localize with the well-developed cytoskeleton of endogenous keratins. These results showed that, in contrast to keratin pairs K5/K14 and K8/K18, the mHa1/mHb4 pair is unable to develop an extensive keratin network on its own and that there are possible differential abilities among these hair keratins and other keratins to form well-developed cytoplasmic networks.

Animals↗
PubMed1996

Accelerated evolution of crotalinae snake venom gland serine proteases.

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

Amino Acid Sequence↗
PubMed1996

Epitope-tagging of a functional thyrotropin receptor: detection of the native receptor on intact cells.

To facilitate immunological detection of thyrotropin receptor (TSHR), we inserted a c-myc epitope within the unique, 50 amino acid segment of the ectodomain (TSHRmyc). When stably expressed in 293 human embryonal kidney (HEK) cells, TSHRmyc demonstrated high affinity TSH binding and the ability to produce cAMP in response to TSH. Binding of the myc monoclonal antibody 9E10 to 293-TSHRmyc cells could be detected with [125I] anti-mouse IgG. No competition was observed between TSH and 9E10 binding to 293-TSHRmyc. Immunoprecipitation by 9E10 of TSHRmyc revealed TSHR forms of approximately 95 and approximately 100 kDa. Endoglycosidase digestion identified the approximately 95 kDa species as the single chain precursor with high mannose carbohydrate. The approximately 100 kDa single chain receptor contained mature, complex carbohydrate. No smaller species of TSHR subunits or proteolytic fragments was observed. Again TSH did not inhibit immunoprecipitation of TSHRmyc by 9E10. These data demonstrate that the normally functioning c-myc epitope-tagged TSHR can be detected directly and in native form with a readily available anti-myc 9E10 and without the need for prior affinity capture. Lack of competition between 9E10 and TSH suggests that at least part of the 50 amino acid segment in TSHR ectodomain is not a TSH binding site. This epitope-tagged TSHR will be valuable for further studies on the synthesis and trafficking of TSHR.

Antibodies, Monoclonal↗
PubMed1996

The loss of recombinant human granulocyte colony-stimulating factor and recombinant human TNF-alpha priming effects on the superoxide-generating response in exudated neutrophils is associated with a decrease in their receptor affinities.

Several cytokines are known to enhance FMLP-stimulated superoxide generation in human circulating blood neutrophils through binding to their specific receptors, a process referred to as the priming effect. The priming effects produced by recombinant human granulocyte CSF (rhGCSF) and TNF-alpha (rhTNF-alpha) on FMLP-stimulated superoxide production in human and rabbit blood neutrophils were compared with their effects in their respective tissue neutrophils, i.e., human salivary and rabbit peritoneal neutrophils. The receptor binding characteristics of rhGCSF and rhTNF-alpha were also compared between the two types of neutrophils. Both rhGCSF and rhTNF-alpha produced dose-dependent priming effects on FMLP-stimulated superoxide production in human blood neutrophils, whereas they failed to produce any priming effects in human salivary neutrophils. Similar results were obtained for the priming effects by rhGCSF in rabbit blood and peritoneal neutrophils. A decrease in receptor binding affinity, but not in receptor density, in tissue neutrophils was demonstrated by analyzing the binding of [125I]rhGCSF and [125I]rhTNF-alpha. These findings suggest that tissue neutrophils are less responsive to rhGCSF and rhTNF-alpha in the modulation of FMLP-stimulated superoxide generation. This is due at least in part to the lower affinities of GCSF and TNF-alpha to their receptors in tissue neutrophils. This marked difference in priming effects by cytokines between blood and tissue neutrophils may represent an early step in the defensive responses against invading microorganisms or Ags.

Animals↗
PubMed1996

Study of paired-pulse inhibition of transcallosal response in the pyramidal tract neuron in vivo.

The effects of a specific GABAB receptor antagonist, p-(3-aminopropyl)-p-diethoxymethyl-phosphonic acid (CGP 35348), on pyramidal tract neuron responses to transcallosal stimulation were investigated in the cat motor cortex in vivo. The paired-pulse method was used to obtain more insight into the role of GABAB receptors. At a 200-ms inter-stimulus interval the spike response was inhibited in 75% of the neurons. There was an approximately 40% depression of the mean spike value in the control. CGp 35348 reduced paired-pulse inhibition, while (-)-baclofen increased it. Stronger drug effects on the second stimulation-induced response possibly indicate their presynaptic action on GABAB receptors.

Animals↗
PubMed1996

Induction and its spread of apoptosis in rat spinal cord after mechanical trauma.

This study was undertaken to examine the spinal cord of rats after trauma for the induction of apoptosis. DNA ladder formation was demonstrated by gel electrophoresis of the DNA obtained from the spinal cord at the site of injury after trauma from 12 h to 8 days and was most significant on Days 3 and 4. As for the longitudinal spread of the injury in the spinal cord, the ladder formation was observed in the two segments adjacent to the site of trauma 3 days after trauma. Histological examination using in situ end labeling also demonstrated the appearance of apoptosis. These results provide the evidence for the induction of apoptosis in the spinal cord after trauma in rats.

Animals↗
PubMed1996

Suppression of inducible nitric oxide synthase mRNA expression by tryptoquinone A.

Triptoquinone A (TQA), which is an anti-inflammatory constituent in plants, was studied for its suppressive effect on nitric oxide production by LPS. TQA significantly suppressed smooth muscle relaxation and increase in cyclic GMP levels by nitric oxide (NO) in an L-arginine-induced relaxation experiment. The mechanistic studies showed that TQA did not directly inhibit NO radicals and inducible nitric oxide synthase (iNOS) enzyme but suppressed IL-1 beta and iNOS mRNA expression by LPS. The suppression level of iNOS gene expression by TQA was comparable to that by dexamethasone. TQA may be a useful candidate for the development of a drug as a potent inhibitor of iNOS gene over-expression.

Animals↗
PubMed1996

Involvement of G protein-coupled receptor kinase 5 in homologous desensitization of the thyrotropin receptor.

Homologous desensitization of G protein-coupled receptors involves agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs). To identify GRK(s) that play a role in homologous desensitization of the thyrotropin (TSH) receptor, thyroid cDNA was amplified by polymerase chain reaction using degenerate oligonucleotide primers from highly conserved regions in GRK family. GRK5 is found in the predominant isoform expressed in the thyroid. Rat GRK5 cDNA was then isolated, which encodes a 590-amino acid protein with 95% homology to human and bovine homologs. Northern blot identified GRK5 mRNA of approximately 3, 8, and 10 kilobases with highest expression levels in lung > heart, kidney, colon > thyroid. In functional studies using a normal rat thyroid FRTL5 cells, overexpression of GRK5 by transfecting the plasmid capable of expressing the sense GRK5 RNA suppressed basal cAMP levels and augmented the extent of TSH receptor desensitization, whereas suppression of endogenous GRK5 expression by transfecting the antisense GRK5 construct increased basal cAMP levels and attenuated the extent of receptor desensitization. Although exogenously overexpressed GRK6 also enhanced TSH receptor desensitization, we conclude that GRK5, the predominant GRK isoform in the thyroid, appears to be mainly involved in homologous desensitization of the TSH receptor.

Amino Acid Sequence↗
PubMed1996

Immunohistochemical detection of Bax and Bcl-2 proteins in gerbil hippocampus following transient forebrain ischemia.

Time-course expression of Bax and Bcl-2 proteins, identified as apoptosis-regulating molecules, was assessed in gerbil hippocampus following transient forebrain ischemia. Brain sections from animals sacrificed at 48, 72, 96 h and 7 days following 5 min ischemia were immunohistochemically evaluated using polyclonal antibodies specific for Bax and Bcl-2 proteins, respectively. The intensity of Bax expression in CA1 neurons increased with time and peaked at 72 h, and immediately disappeared at 96 h following 5 min ischemia. No expression of Bcl-2 in the CA1 neurons was recognized in all the time evaluated.

Animals↗
PubMed1996

Deficiency of the Hck and Src tyrosine kinases results in extreme levels of extramedullary hematopoiesis.

Expression of the Src-family kinases--Src, Hck, and Fgr--increases dramatically during myeloid cell development. Src-deficient mice exhibit functional abnormalities in only one myeloid cell type, the osteoclast, resulting in impaired bone remodeling and osteopetrosis, while hck-/- or fgr-/- mice have few and subtle myeloid cell deficiencies. To determine whether these limited phenotypes are due to the coexpression of multiple Src-family kinases with overlapping functions, we have intercrossed src-/- mice to hck-/- and fgr-/- mutants to produce double mutants. Two thirds of hck-/- src-/- double mutants die at birth; surviving animals develop a severe form of osteopetrosis, resulting in extreme levels of splenic extramedullary hematopoiesis, anemia, and leukopenia. These hematopoietic defects are caused by abnormalities in the bone marrow environment because hck-/- src-/- mutant stem cells reconstitute a normal hematopoietic system in irradiated wild-type mice. In contrast, fgr-/- src-/- double mutants have no defects beyond those observed in src-/- animals. Cultured normal murine osteoclasts express abundant amounts of Src, Hck, and Fgr and Hck levels are increased in src-/- osteoclasts. These observations suggest that Hck and Src serve partially overlapping functions in osteoclasts and that the expression of Hck in src-/- osteoclasts ameliorates their functional defects.

Animals↗
PubMed1996

Effects of exogenous transforming growth factor-beta 1 on spinal cord injury in rats.

This study was undertaken to examine the effect of transforming growth factor-beta 1 (TGF-beta 1) administered into the subarachnoid space after spinal cord injury (SCI) on the increased production of inducible-nitric oxide synthase (i-NOS) in the injured spinal cord in rats. The expression of i-NOS mRNA after SCI was remarkably down-regulated by TGF-beta 1 in vivo. Rats treated with TGF-beta 1 showed a better outcome regarding hindlimb motor dysfunction in the first 5 days after injury compared to the saline-treated rats. However, the final outcome was not better and fibrous scar formation in the injured spinal cord was more evident, which was demonstrated as increased immunoreactivity of fibronectin in the later stage after SCI. These results provide evidence of both positive and negative contributions of TGF-beta 1 to the pathology associated with SCI.

Animals↗
PubMed1996

Inducible nitric oxide synthase in uterine smooth muscle.

The expression of inducible nitric oxide synthase (iNOS) mRNA in rat uterus upon in vivo stimulation with lipopolysaccharide (LPS) was studied by reverse transcription and polymerase chain reaction. The injection of LPS induced an increase in mRNA levels of a macrophage-type iNOS. In unstimulated rats, low levels of iNOS mRNA was detected in the uterus and lungs, but absent or negligible in the kidneys and liver. NO was produced in the LPS-treated uterus by addition of 1 to 1000 microM L-arginine. The production of NO in uterine tissue that faces the outside of the body may provide a bacteriocidal protective function against microorganisms in physiological condition. However, NO produced in a large amounts by cytokine and LPS may play some pathological reaction during septic shock or infection.

Animals↗
PubMed1996

Roles of nitric oxide in compression injury of rat spinal cord.

Nitric oxide (NO) was measured directly after spinal cord injury (SCI) in rats by an ESR spin-trapping technique using Fe2+ and diethyldithiocarbamate (DETC). The levels of NO and lipid peroxides expressed as thiobarbituric acid reactive substances (TBARS) were increased by SCI in the injured region and the adjacent central region. Pretreatment with 30 mg/kg of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase, accelerated increases of the TBARS level and myeloperoxidase (MPO) activity in the injured tissue and caused deterioration of hind limb motor function after SCI, suggesting that NO formation by constitutive NO synthase (c-NOS) has a protective effect against cellular damage resulting from ischemia-reperfusion after SCI. Though c-NOS mRNA expression was not altered after SCI, inducible NO synthase (i-NOS) mRNA expression increased to a maximum of 24 h after SCI with progress of motor dysfunction. Intravenous injection of L-NAME (0.1 mg/kg) 6, 24, 48, and 72 h after SCI reduced the motor disturbance. These results indicate that NO induced by i-NOS may be neurotoxic in the subacute phase after SCI.

Animals↗
PubMed1996

Effect of dietary fat content on oral bioavailability of menatetrenone in humans.

The purpose of this investigation was to determine the effects of fat content and frequency of meals on the oral bioavailability of menatetrenone (2-methyl-3-all-trans-tetraprenyl-1,4-naphthoquinone), a vitamin K2 with four isoprene units. In the first series of studies, menatetrenone (15 mg) was administered at breakfast time to 18 healthy male volunteers after meals with three different fat contents (meals A, B, and C) on three occasions in a crossover design. The three types of meals had almost the same calorie content (721-746 kcal) with varied fat contents (A, 8.8 g; B, 20.0 g; C, 34.9 g). The area under the plasma menatetrenone concentration-time curve within the first 24 h (AUC0-24) increased with increase of fat content: 371 +/- 194, 485 +/- 150, and 1024 +/- 341 ng.h/mL (mean +/- SD, n = 18) after meals A, B, and C, respectively. On the fourth occasion, the same dose of menatetrenone was administered to all volunteers after taking meal B, but in this case the lunch 5 h after drug administration was omitted from the protocol. The time profile of plasma menatetrenone showed a single peak when lunch was not taken, whereas it showed two peaks with lunch. On the fifth occasion, 12 out of 18 volunteers took the same dose of menatetrenone after a meal with the highest fat content (53.8 g of fat and 789 kcal; meal D), showing that AUC0-24 was almost the same as that for meal C, 1027 +/- 389 and 991 +/- 392 ng.h/mL (n = 12) for meals C and D, respectively. The oral bioavailability of lipid-soluble vitamin K was influenced by the fat content of a meal, although the increase in bioavailability seemed to reach a peak when the lipid content of the meal was > 35 g.

Administration, Oral↗