Biomedical subjects

N Yamaguchi

Publications and source records attributed to N Yamaguchi.

At least 217 records · Page 12Linked to original sources

PubMed1997

Purification, cDNA cloning, and characterization of a new serpin with megakaryocyte maturation activity.

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

Amino Acid Sequence↗

PubMed1997

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.

Animals↗

PubMed1997

A new method for estimating spinal cord function. Refractory period of conductive spinal cord evoked potentials.

STUDY DESIGN: This study was designed to examine the possibility of a new spinal cord monitoring method using measurement of the refractory period to monitor spinal cord function. OBJECTIVES: To determine whether measuring the refractory period and the recovery rate of conductive spinal cord evoked potential is a useful method for estimating spinal cord function. BACKGROUND: Measuring the refractory period and constructing the recovery curve have been used to investigate peripheral nerve function. Spinal cord evoked potential elicited by the single stimulus usually is used to evaluate spinal cord function, and it has been said that 50% attenuation of the amplitude is the critical alarm level. METHODS: In anesthetized cats, amplitude, area, and latency were measured on a personal computer from subtracted data collected with a paired-stimulation technique. The authors constructed recovery curves of ascending and descending conductive spinal cord evoked potentials and measured the refractory period during spinal cord compression. RESULTS: When the amplitude of the ascending spinal cord evoked potential began to decrease during spinal cord compression, the amplitude of the response elicited by the second stimulus with interstimulus intervals of 0.8 msec and 1.0 msec decreased more significantly. When the amplitude of the ascending spinal cord evoked potential decreased to 50% of the precompression amplitude, the mean value of the absolute refractory periods of the ascending and descending spinal cord evoked potentials became prolonged from 0.40 +/- 0.007 msec to 0.53 +/- 0.014 msec, and the mean values of their amplitude and area recovery rates decreased from 75% +/- 1% to 35% +/- 2% (interstimulus interval, 0.8 msec) and from 81% +/- 1% to 46% +/- 2% (insterstimulus interval, 1.0 msec). CONCLUSIONS: The change of the responses elicited by the paired stimuli is more sensitive than those elicited by the single stimulus in the spinal cord evoked potentials. The absolute refractory periods and the recovery rate during 50% attenuation of the precompression amplitude is the critical alarm level in spinal cord monitoring.

Animals↗

PubMed1997

Polymorphism of murine Fas ligand that affects the biological activity.

Fas ligand (FasL) is a member of the tumor necrosis factor family and induces apoptosis in Fas (CD95)-bearing target cells. In this study, we generated several mAbs that react with mouse FasL (mFasL) and characterized their functional properties. One of these mAbs, K10, specifically reacted with mFasL derived from C57BL/6 (B6) mice, but not that from BALB/c mice as estimated by surface staining and blocking of cytotoxic activities of mFasL transfectants, suggesting a polymorphism of mFasL. Sequence analysis of mFasL cDNA from several strains revealed that BALB/c and DBA/2 mice have three nucleotide differences from the known B6 and C3H sequences, which result in two amino acid substitutions (Thr-184 --> Ala-184 and Glu-218 --> Gly-218) in the extracellular region. Analysis of the K10 reactivity and genotyping by PCR-restriction fragment length polymorphism revealed that inbred mice segregate into the following two allotypes: mFasL.1 (B6, C3H, MRL, SJL, NOD, NZB, NZW) and mFasL.2 (BALB/c, DBA/1, DBA/2). Interestingly, COS7 cells expressing BALB/c FasL lysed Fas-bearing target cells more efficiently than those expressing B6 FasL. Furthermore, BALB/c-derived CD8-FasL fusion protein, which is composed of the extracellular domains of human CD8alpha and mFasL, exhibited 9-fold higher specific activity than did B6-derived CD8-FasL. These results suggest that in mFasL.2 mice the Fas/FasL system works more effectively than in mFasL.1 mice.

Alleles↗

PubMed1997

Deletion map of chromosome 9 and p16 (CDKN2A) gene alterations in neuroblastoma.

We reported previously that loss of heterozygosity (LOH) on chromosomes 2q, 9p and 18q frequently occurs in neuroblastoma and that patients with 9p LOH in the tumors showed statistically significant association with an advanced stage of the disease and poor prognosis. To determine the role of chromosome 9 loss in neuroblastoma, we performed deletion mapping of chromosome 9 in 80 cases of neuroblastoma using 11 polymorphic microsatellite markers and a restriction fragment length porymorphism marker. LOH at one or more loci on chromosome 9 was detected in 33 of 80 cases (41%). Chromosome 9p was lost in 24 of 80 cases (32%), whereas chromosome 9q was lost in 18 of 80 cases (23%). There were two commonly deleted regions mapped to 9p21 between the D9S171 marker and the IFNB1 marker and 9q34-qter distal to the D9S176 marker. In addition, patients with LOH at 9p21 but not at 9q34-qter in the tumors showed statistically significant association with poor prognosis (P = 0.023). Because the commonly deleted regions at 9p21 includes the p16 (CDKN2A) gene, the status of the p16 gene was further examined in 80 fresh tumors and 19 cell lines of neuroblastoma. A missense mutation was detected at codon 52 in a fresh tumor. The p16 gene was not expressed in 13 of 19 cell lines (72%), and 5 of the 13 cell lines displayed methylation of the CpG island surrounding the first exon of the p16 gene. These results suggest that the p16 gene is a candidate tumor suppressor gene for neuroblastoma, and its inactivation may contribute to the progression of neuroblastoma.

Carrier Proteins↗

PubMed1997

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Animals↗

PubMed1997

Genomic structure and promoter analysis of the gene encoding MM3, a member of transmembrane 4 superfamily.

We have isolated genomic clones encoding hamster MM3, a member of transmembrane 4 superfamily (TM4SF). Nucleotide (nt) sequence analysis revealed that it is composed of 5 exons spanning about 8 kb. The exon-intron organization of the MM3 gene was quite different from those of other TM4SF members. We also identified its transcription start points (tsp) and the promoter region. Deletion analysis of the promoter revealed that about 160-bp region containing TATA-box, CAAT-box and GC-box was necessary for efficient transcription in cultured cells.

Amino Acid Sequence↗

PubMed1997

Electroencephalographic abnormalities in patients with presenile dementia of the Alzheimer type: quantitative analysis at rest and during photic stimulation.

In the present study, quantitative electroencephalographic (EEG) analysis was performed at rest and during photic stimulation (5, 10, and 15 Hz) in nine patients with presenile dementia of the Alzheimer type (AD; mean age at onset, 55 years) and nine sex- and age-matched control subjects. Compared with the normal controls, the AD patients had a significantly lower alpha-2 and beta band power in the resting EEG as well as a significant increase in delta and theta band power. EEG analysis during the photic stimulation demonstrated that the AD patients had a significantly lower EEG power during photic stimulation for the alpha (9.8-10.2 Hz) and beta bands (14.8-15.2 Hz) corresponding to photic stimulation at 10 Hz and 15 Hz, respectively. In addition, when we examined EEG changes from rest to the stimulus condition, the AD patients were found to show significantly smaller changes in EEG power mainly over the posterior regions, irrespective of the stimulus frequency. These findings provide evidence that AD patients have EEG abnormalities in both non-stimulus and stimulus conditions, and suggest diminished EEG reactivity to photic stimulation.

Alzheimer Disease↗

PubMed1997

Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain.

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.

Adenocarcinoma↗

PubMed1997

Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits.

An experimental model of early-stage cardiomyopathy was created by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta-adrenoceptors or M2-muscarinic receptors. Thirty male rabbits were used and divided into three groups: a control group (n = 10), a group immunized with the peptide corresponding to the beta-adrenoceptor (beta 1 group) (n = 10) and a group immunized with the peptide corresponding to the M2-muscarinic receptor (M2 group) (n = 10). If the sera from both groups of immunized rabbits high-titres of anti-peptide antibodies were found throughout the study period but not in the sera from control rabbits or in the preimmune sera of immunized rabbits. No significant cross-reaction with peptides other than those used for immunization was found. The myocardial receptor density of both immunized groups displayed a strong trend toward receptor up-regulation. This was significant in the beta 1 group but not in the M2 group. Both groups of immunized rabbits displayed significantly enlarged ventricles and thinner walls, as compared with the control group. However, in contrast to the beta 1 group, which showed enlarged cavities in both left and right ventricles, the M2 group was mainly affected in the right ventricles. Moreover, morphological examinations of the hearts of rabbits from both immunized groups demonstrated focal myofibrillar lysis, loss of myofilament, mitochondrial swelling and condensation, sarcoplasmic vacuolation, deposition of dense granules in the sarcoplasm and the myofibrils. One of the sex control rabbit hearts which were examined showed mild degenerative changes in the myocardium and scant mononuclear cell infiltration. However, when all the control rabbit hearts were examined by electron microscopy, no significant alterations were found. These results suggest that immunization by peptides, corresponding to the target sequences for anti-receptor autoantibodies in idiopathic dilated cardiomyopathy, induces morphological changes in the heart similar to those found in the human disease.

Amino Acid Sequence↗

PubMed1997

Differences in enhancing effects of zolpidem and benzodiazepine drugs on recurrent inhibition in rat hippocampal slices.

It has been reported that the clinical and electroencephalographic profiles of zolpidem, a non-benzodiazepine drug which binds preferentially to the omega 1 benzodiazepine recognition sites located within the GABAA receptor complex, are different from those of benzodiazepine drugs, which bind non-selectively to the omega 1 and omega 2 sites. In order to clarify the electrophysiological mechanism underlying the unique profile of zolpidem, the present study compared the enhancing effects of zolpidem and two benzodiazepine drugs, triazolam and diazepam, on recurrent inhibition. This inhibition was expressed as suppression of the orthodromically induced population spikes by the preceding antidromic stimulation of the alveus in the CA1 region of rat hippocampal slices. The rank order of potency for enhancing recurrent inhibition was triazolam > diazepam > zolpidem. From the present results and previously reported findings that zolpidem has a lower affinity for the omega 2 sites than diazepam while both have the same affinity for the omega 1 sites, we concluded that the hippocampal recurrent inhibition appears to be enhanced mainly by activation of the omega 2 sites, but not by that of the omega 1 sites. Furthermore, the lower potency of zolpidem for enhancing recurrent inhibition may underlie its unique profile in terms of its clinical and electroencephalographic effects.

Animals↗

PubMed1997

Reflex adrenal medullary secretion during coronary occlusion mediated by cardiac receptors with afferent vagal fibres in the rat.

The relative contribution of ventricular receptors and sinoaortic baroreceptors to the reflex release of adrenaline upon coronary occlusion was studied in anaesthetised rats submitted to a 30-min occlusion of the left coronary artery. Arterial adrenaline concentrations, heart rate and blood pressure were evaluated in control and sham-operated rats, and following sinoaortic denervation (SAD), bilateral vagotomy with and without SAD, ventricular application of lidocaine, neonatal treatment with capsaicin and cervical section of teh spinal cord. In all groups submitted to coronary ligation, blood pressure decreased sharply upon ligation and remained significantly reduced throughout occlusion while heart rate changes were not significant. In control rats, arterial adrenaline concentrations were significantly increased 15 and 30 minutes following coronary occlusion. The increase in adrenaline concentrations observed upon coronary ligation following SAD was not different from that of the control group. On the other hand, vagotomy with or without SAD, ventricular application of lidocaine, neonatal capsaicin treatment and spinal section all greatly reduced the increase in plasma adrenaline values. These results suggest that a large part of the reflex release of adrenaline upon coronary occlusion is mediated by ventricular receptors with unmyelinated vagal afferent fibres.

Adrenal Medulla↗

PubMed1997

Human cytomegalovirus open reading frame UL11 encodes a highly polymorphic protein expressed on the infected cell surface.

Human cytomegalovirus (HCMV) open reading frame (ORF) UL11 locates within a polymorphic region of the viral genome identified previously by a restriction-fragment-length-polymorphism. We report here that ORF UL11 encodes a polymorphic protein expressed on the surface of HCMV-infected cells. First, we determined the nucleotide sequence of ORF UL11 from ten strains and compared it among the strains. Out of 205 amino acids consisting of the predicted N-terminal region beside the putative transmembrane stretch in strain AD169, 88 residues were divergent on more than one strain. In contrast, the predicted C-terminal side including the putative transmembrane domain was identical at the amino acid sequence level. In addition, the number and location of predicted cysteine residues were also conserved. Next, we screened a cDNA library from HCMV-infected cells and obtained a cDNA clone containing the full-length ORF UL11. Finally, we identified the gene product of UL11 on the surface of HCMV-infected cells by FACS analysis with polyclonal antibodies generated against a glutathione S-transferase/UL11 fusion protein. The fusion protein contained a region within the N-terminal side next to the predicted transmembrane stretch. These results indicate that the N-terminal side of UL11 protein containing variable amino acid residues protrudes from the infected cell surface.

Amino Acid Sequence↗

PubMed1997

Effects of spinal cord ischemia on the refractory period of descending spinal cord evoked potential.

The recovery cycle, following the conduction of action potentials along a nerve fiber, consists of the absolute refractory period (ARP), the relative refractory period (RRP) and the supernormal period (SNP). The recovery cycle of the descending conductive spinal cord evoked potential (SCEP) was shown during normal state, ischemia and after ischemia using paired stimuli in cats. During ischemia the refractory period revealed a trend towards increment. Five minutes after reperfusion the refractory period decreased transiently compared with the normal level and within 30 min the refractory period returned to the normal level. The recovery curve of the 2nd potential showed different pattern compared with that of the 1st potential. Moreover, during ischemia, firstly the 3rd potential and secondly the 2nd potential of the SCEP elicited by the 2nd stimulus were disturbed. These results demonstrated that there is increased excitability of the spinal cord to the second stimulus after a brief period of ischemia, and that the 2nd and 3rd potentials are evoked synaptically and easily disturbed during ischemia. Measuring the SCEP elicited by paired stimuli or constructing the recovery curve of the SCEP is useful for the electrophysiological assessment of spinal cord function.

Action Potentials↗

PubMed1997

Prediction of consistency of meningiomas with preoperative magnetic resonance imaging.

BACKGROUND: The consistency of a meningioma is one of the important factors in determining the surgical outcome. If the surgeon is aware of the consistency of a meningioma preoperatively, the surgical plans will be influenced. A few papers have described the correlation between consistency of meningiomas and their magnetic resonance imaging (MRI) findings. However, prediction of consistency with MRI is still difficult. We have tried to predict the consistency of meningiomas with MRI findings more precisely. METHODS AND RESULTS: Fifty patients diagnosed as having intracranial meningiomas were studied with 1.5 Tesla MRI. We compared the MRI findings with tumor consistency. The intensities of the tumors were categorized into three grades (low, iso, and high) compared to that of the gray matter. T1-weighted images had no specifics, but T2-weighted images and proton density images were useful for the prediction of tumor consistency. Hyperintensity on protein density (PD) and T2-weighted images was a sign of a soft tumor. CONCLUSION: We presume that T2 and PD are useful for predicting consistency of meningiomas, and their water content is one of the main factors in their consistency. Histology may be one of the factors helpful in defining the consistency of a tumor. In this series, we found no relationship between histology and MRI findings, nor between histology and consistency. If the meningioma is believed to be hard, preoperative endovascular embolization is beneficial, which will induce necrosis of the meningioma and make it soft enough to be removed more easily and safety.

Adult↗

PubMed1997

Radioimmunoassay of meterelin and pharmacokinetics after single injection and implant administration in dogs.

A sensitive and specific radioimmunoassay for a novel luteinizing-hormone-releasing-hormone (LHRH) agonist, [2-Me-D-Trp6, DesGly10]LHRH ethylamide (meterelin), was developed for documenting the pharmacokinetic parameters of this peptide following its intravenous (iv) and subcutaneous (sc) administration in dogs. The assay was also used for monitoring meterelin in plasma following its release from d,l-lactide-glycolide implants in the same species. Rabbit antisera generated against [DespyroGlu1] meterelin and conjugated to bovine serum albumin with glutaraldehyde showed high specificity, whereas crossreactivity to LHRH and its fragments and to analogs with substitutions at residues 6 and 10 was found insignificant. The assay was validated in terms of accuracy (recovery range, 94.0-105.4%), in terms of precision (intra- and interassay variations of 10.0-12.4% and 8.6-11.3%, respectively), and in terms of sensitivity (minimum detectable dose of 2.7 pg/assay). Following iv acute administration, a biexponential decline of plasma meterelin levels was observed, with distribution and elimination half-lives of 5.9 +/- 2.5 and 106 +/- 22 min, respectively. After sc acute administration, the elimination half-life was in the range of 103 to 173 min. The systemic clearance (CLT) ranged from 1.6 to 2.6 mL/min/kg, and the volume of distribution at steady state (Vdss) varied from 285 to 438 mL/kg. The elimination half-life (T1/2 beta), Vdss, and ClT were not significantly different after both routes of administration over the 1-100-microgram/kg dose range of peptide studied. Castrate levels of testosterone were attained 10 days after sc administration of the implant, lasted for up to 247 days, and were well correlated with plasma levels of meterelin.

Amino Acid Sequence↗

PubMed1997

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.

Animals↗

PubMed1997

Isoforms of gp138, a cell-fusion related protein in Dictyostelium discoideum.

Sexual development of Dictyostelium discoideum is a unique and useful system for the study of sexual phenomena. We have been studying molecular mechanisms of sexual cell fusion in D. discoideum and have identified several relevant cell-surface proteins. One of the proteins, gp138, was identified as a target molecule for fusion-blocking antibodies, and two genes for gp138, GP138A and GP138B, were cloned. The participation of gp138 in the sexual cell fusion was confirmed by antisense RNA mutagenesis, but it is unclear which of the genes encodes gp138. Moreover, the presence of a third gene for gp138 was indicated by gene disruption. In the present study, we generated strains of D. discoideum overexpressing either GP138A or GP138B to investigate the products of these genes. The transformants overexpressing GP138A and GP138B overproduced glycoproteins with molecular masses of 135 and 130 kDa, respectively. Although their molecular masses were different from that of gp138, the results of peptide mapping and amino acid sequencing showed that they are related to proteins, suggesting that the proteins encoded by GP138A and GP138B are isoforms of gp138 protein.

Amino Acid Sequence↗