Biomedical subjects
Publications and source records attributed to Y Murakami.
We report here cloning and sequencing human antizyme inhibitor from a human kidney cDNA library. Amino acid sequence deduced from the nucleotide sequence shows 92.9% identity to that of rat antizyme inhibitor. Northern blot analysis reveals that antizyme inhibitor is expressed in human liver.
While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.
Addition of spermidine to culture medium of insect cells, Trichoplusia ni 5, at a low cellular density suppressed ornithine decarboxylase (ODC; EC 4.1.1.17) activity and induced ODC inhibitory activity. The inhibitory factor was non-dialyzable, temperature-sensitive, and could reversibly form an inactive complex with ODC. It showed a time-independent and non-stoichiometric pattern of inhibition. Upon addition of spermidine to cultured cells with preinduced ODC, the enzyme decayed more rapidly than after addition of cycloheximide. These data strongly suggested that ODC of Tn5 cells is under negative feedback control by polyamines, in which an antizyme-like regulatory protein plays an essential role.
Transcardiac serotonin differences in nonischemic patients with endothelial dysfunction significantly increased in the early morning. Platelet secretion could occur without coronary stenosis.
BACKGROUND: Recent studies indicated that ischemic microvascular damage may be reversible or progressive after coronary reflow. Intramyocardial hemorrhage is a phenomenon that reflects severe microvascular injury. We examined the relationship between temporal changes in microvascular perfusion patterns detected by myocardial contrast echocardiography (MCE) and intramyocardial hemorrhage detected by magnetic resonance imaging (MRI) in patients with acute myocardial infarction (AMI). METHODS AND RESULTS: The study population consisted of 24 patients with anterior AMI. All patients underwent MCE shortly after reflow and in the chronic stage (a mean of 31 days after reflow). Wall motion score (WMS) was determined as the sum of 16 segmental scores (dyskinetic/akinetic=3 to normal=0) at days 1 and 31. Gradient-echo acquisition and gadolinium-DTPA-enhanced spin-echo MRI were performed within 10 days after reflow. In MCE shortly after reflow, 16 patients (67%) showed contrast enhancement and the other 8 patients (33%) showed a sizable contrast defect. In the chronic stage, a persistent contrast defect was observed in 7 of 8 patients with a contrast defect shortly after reflow. Consistent contrast enhancement was observed in 12 of 16 patients (75%) with contrast enhancement shortly after reflow, indicating that a contrast defect newly appeared in 4 patients (25%). Intramyocardial hemorrhage was detected in 9 patients (38%): 5 of 7 patients with a persistent contrast defect and in all 4 patients with a new appearance of a contrast defect during the chronic stage. The patients without hemorrhage showed a significant improvement in WMS compared with patients with hemorrhage at day 31 (5+/-5 versus 19+/-6, P<.0005). CONCLUSIONS: These results suggest that irreversible microvascular damage to the ischemic myocardium may cause intramyocardial hemorrhage after reflow, associated with impaired recovery of left ventricular function. Contrast enhancement within the risk area shortly after reflow does not necessarily indicate long-term microvascular salvage.
Na,K-ATPase alpha subunit has three isoforms whose expression is regulated developmentally and hormonally. Na,K-ATPase alpha3 subunit gene (Atpla3) is expressed only in brain and neonatal heart in a rat. The purpose of this study is to analyze cis-acting elements and trans-acting factors regulating the transcription of Atpla3 in cultured neonatal rat cardiocytes. Transient transfection assays with Atpla3-luciferase chimeric construct and a series of 5' sequential deletion mutations revealed the existence of positive regulatory elements from -74 to -59 and from -59 to -39. A factor was identified to bind across -59 by gel retardation assay. Methylation interference and DNase I footprinting analyses revealed the binding region from -74 to -53 (positive regulatory element (PRE) 1). The binding factor was identified to be NF-Y by gel retardation assay using specific antibody. Gel retardation and methylation interference analyses revealed that factors bind to two other elements from -54 to -43 (PRE2) and from -25 to -13 (PRE3). The binding factors were identified to be Sp1/Sp3 using specific antibodies. The functions of above-mentioned three elements were examined by transient transfection assay with various combinations of mutations. They all regulated the transcription positively and a synergistic enhancement of it was observed. Roles of NF-Y in the transcriptional activation and synergy are discussed.
Immunoreactive galanin and galanin message associated peptide (GMAP) were detectable in rat hypothalamus in the concentration of 563 +/- 23 and 14.3 +/- 3.1 fmol/hypothalamus, respectively. gamma-Aminobutyric acid (GABA) elicited a dose-related increase in galanin release from rat hypothalamic fragments, which was inhibited by picrotoxin, a GABA antagonist. Growth hormone (GH) secretion from rat anterior pituitary cells were stimulated by rat galanin, but not by GMAP. These findings suggest that hypothalamic galanin, but not GMAP, may play roles in GH secretion induced by GABAergic mechanisms in the rat.
Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographical and optical images of human chromosomes using a sharp and bent optical fiber as a near-field optical probe. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. The SNOAM system does not need pretreatment of samples such as metal coating or chemical immobilization. Near-field topographic and fluorescence images provided useful information on native chromosome structure.
We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RB1) locus on chromosome 13q14 in 20 non-small-cell lung cancers (NSCLCs) using polymorphic markers. The expression of RB protein was examined by immunohistochemical analysis of paraffin-embedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the RB1 locus, whereas only 2 of the 10 tumors lost expression of the RB protein. These 2 tumors had mutations in the remaining RB1 allele. Thus, inactivation of the RB1 gene appears to be involved in a small subset of NSCLCs only. To elucidate the presence of tumor-suppressor genes other than RB1 on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at one locus, and the regions 13q12.1-qter, 13q12.2-14.2 and 13q14.1-q14.3, including the RB1 locus, were deleted in significant numbers of the tumors. Our results suggest that, in addition to the RB1 gene, abnormalities of other tumor-suppressor genes on chromosome 13q are involved in the development of human NSCLCs.
Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.
To clarify the mechanism of brain and spinal cord impairment in Rett syndrome (RS), we measured the cerebrospinal fluid (CSF) levels of substance P in 20 patients with RS including 16 childhood patients and 4 adult patients. Findings were compared with those obtained in age-matched controls and diseased controls. The CSF level of substance P was significantly lower in patients with RS compared with controls. The alteration in the CSF level of substance P may be related to the neurological impairment, especially autonomic dysfunction, and neuropathological involvement of dorsal root ganglia and peripheral nerve observed in RS.
We examined the clinical features of 14 men (mean age 72 years) with postoperative enteritis caused by methicillin-resistant Staphylococcus aureus (MRSA). The patients had all undergone surgery for the treatment of digestive diseases and had received antibiotic prophylaxis consisting of an extended-spectrum cephem. Diarrhea appeared a mean of 3.3 days postoperatively and lasted for 5 days on average. In severe cases organ insufficiency was involved. Coagulate-positive staphylococci were the predominant organisms isolated from watery diarrhea. In 13 of 14 patients, coagulase type II isolates producing enterotoxins A, C and toxic shock syndrome toxin-1 (TSST-1) with enterotoxin A, C, and 1st genes were isolated. These strains were sensitive to vancomycin and arbekacin; however, they were highly resistant to many other antibiotics. We also investigated the effects of a glucocorticoid hormone and gamma globulin on production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) obtained from healthy volunteers. TNF-alpha and IL-2 production was enhanced by TSST-1 and the supernatant of Iscove-modified dulbecco medium, in which coagulase type II isolates producing enterotoxins A, C and TSST-1 with enterotoxin A, C were cultured for 24 h. Both glucocorticoid hormone and gamma globulin suppressed TNF-alpha and IL-2 production, thus suggesting that these drugs may be effective in treating postoperative MRSA enteritis.
A 29-year-old woman was hospitalized with a 1-month history of postprandial epigastric pain, nausea, and vomiting. An upper gastrointestinal tract X-ray series showed a marked narrowing of the pyloric region. A histological examination of duodenal mucosal biopsy samples showed granulomatous inflammation, and thus a diagnosis of intrinsic duodenal Crohn's disease was made. A second upper gastrointestinal tract X-ray revealed a persistent gastric outlet obstruction. At laparotomy, the duodenal wall was found to be thickened over a distance measuring 3.5 cm in length from the pyloric ring. A longitudinal incision was made over the entire length, up to 5.5 cm beyond the pyloric ring on either side, while Finney-type anastomosis was also performed. A postoperative upper gastrointestinal tract X-ray showed an improvement in the gastroduodenal passage. Enteral nutrition therapy was provided postoperatively. Omeprazole was administered at a dose of 20 mg/day for 2 months. The patient currently remains on maintenance therapy with famotidine at 20 mg/day and is clinically doing well.
This study was designed to determine the influence of ileal pouch capacity and anal sphincteric function on the clinical outcome after ileal pouch-anal anastomosis. A total of 24 patients who had undergone ileal pouch-anal anastomosis (J pouch) for ulcerative colitis were studied. The 24-hour stool frequency was found to be inversely correlated with the sensitivity threshold volume (STV), maximal tolerance volume (MTV), and distensibility, but was independent of the maximal resting pressure and maximal squeeze pressure. Patients experiencing nocturnal fecal incontinence had maximal resting pressures that were significantly lower than those of nocturnally continent patients. Among the patients with fecal incontinence, those with frequent soiling had lower resting pressures, STV, and distensibility than the patients with intermittent spotting. In addition, the STV in patients needing nocturnal evacuation were lower than those of patients who did not evacuate after falling asleep. The conclusions are as follows. Both stool frequency and the need for nocturnal pouch evacuation correlated directly with pouch volume. Anal incontinence was more common in patients with low internal sphincteric function. In addition, frequent and gross nocturnal incontinent patients demonstrate a worse function in both the anal sphincter and reservoir than those with intermittent spotting.
The eighth nerve compound action potential (CAP) in 95 guinea pigs was measured using click stimuli to investigate age-related changes in their neural auditory thresholds. The animals were separated into three groups: group A (n = 43, 86 ears; 2-4 months old); group B (n = 29; 58 ears, 13-15 months old); and group C (n = 23; 46 ears, 23-25 months old). With increasing age, a gradual elevation of CAP thresholds was clearly seen among the three groups. The negative peak (N1) latencies of the CAP were prolonged, and the N1 amplitudes of the CAP decreased. There were significant differences in N1 latencies among the three groups and in N1 amplitudes between groups A and B, and between groups A and C. However, the rate of decline of the thresholds as well as the input-output function curves of the CAP varied in some of the oldest animals, suggesting that there were some individual differences in degenerative aging processes of the auditory system.
Intracerebroventricular (i.c.v.) administration of bombesin (0.3 nmol) increased plasma levels of both adrenaline and noradrenaline in urethane anesthetized rats. These bombesin-induced increases were inhibited by i.c.v. pretreatment with pyrilamine, an H1-receptor antagonist. Ranitidine, an H2-receptor antagonist also inhibited the increase of adrenaline, however, its effective dose was much larger than that of pyrilamine. Furthermore, the bombesin-induced increase of noradrenaline was not effectively inhibited by ranitidine. In the next series, turnover of histamine was assessed by measuring accumulation of tele-methylhistamine (t-MH), a major metabolite of brain histamine. I.c.v. administration of bombesin (0.3-3 nmol) increased turnover of hypothalamic histamine, while its intravenous administration was without effect. The present results suggest that the bombesin-induced central activation of sympatho-adrenomedullary outflow is probably, at least in part, mediated through brain histaminergic neurons.
The alpha2-adrenoceptor agonist clonidine, as well as 5-HT2 receptor antagonists, reportedly suppress 5-HT2 receptor-mediated head-twitch behavior. We investigated the effect of alpha2-adrenoceptor antagonists on the suppressive action of 5-HT2 receptor antagonists in mice pretreated with the noradrenaline toxin 6-hydroxydopamine (6-OHDA) or the 5-HT synthesis inhibitor p-chlorophenylalanine (p-CPA). In normal mice, idazoxan (0.08-0.2 mg/kg, IP) or yohimbine (0.2-2.0 mg/kg, IP), both alpha2-adrenoceptor antagonists, had no effect on the head-twitch response caused by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT; 16 mg/kg, IP), but idazoxan significantly enhanced the response at 0.5 mg/kg. On the other hand, these alpha2-adrenoceptor antagonists, at doses that had no effect on the basal number of head-twitches (idazoxan 0.2 mg/kg and yohimbine 0.5 mg/kg), significantly attenuated not only the suppressive effect of clonidine (0.01 mg/kg, IP) on head-twitch response but also that of the 5-HT2 receptor antagonist ritanserin (0.03 mg/kg, IP). Moreover, idazoxan (0.2 mg/kg) also significantly reversed the inhibition by 0.01 mg/kg (IP) ketanserin, a selective 5-HT2 receptor antagonist. Pretreatment with 6-OHDA plus nomifensine but not with p-CPA significantly attenuated the effect of idazoxan (0.2-0.5 mg/kg) on the ritanserin inhibition of the head-twitch response. Prazosin, an alpha1-adrenoceptor antagonist, dose-dependently suppressed the response, and the effect of prazosin (1.25 mg/kg) was significantly attenuated by 0.5 mg/kg idazoxan. These results indicate that endogenous noradrenaline is involved in the apparent antagonistic interaction between selective alpha2-adrenoceptor antagonists and 5-HT2 receptor antagonists in the head-twitch response, and suggest that noradrenaline stimulation of alpha1-adrenoceptors may be involved in this apparent antagonism.
The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on ion channels were examined in GH3 cells human pituitary adenoma cells. In GH3 cells, PACAP-38 (10-9 M) reversibly activated tetrodotoxin-sensitive NA+ channels but had little effect on nicardipine-sensitive Ca2+ channels. PACAP-induced increase in Na+ currents was inhibited by PACAP (6-38), a specific PACAP receptor antagonist, and Rp-cAMPs, an inhibitor for protein kinase A, and mimicked by 8-bromo-cAMP. In human pituitary adenoma cells, PACAP also activated tetrodotoxin-sensitive Na+ channels and growth hormone secretion. These results suggest the possibility that PACAP can activate voltage-gated Na+ channels via adenylate cyclase-protein kinase A pathway in the pituitary.