Biomedical subjects
Publications and source records attributed to A Wada.
8,16-Ethanoretinal 3 was synthesized from 2-methylcyclohexanone. From the binding experiments of 3 and 8,18-ethanoretinal 2 with apoproteins of sensory rhodopsin I and phoborhodopsin, it was found that retinal was incorporated as 6s-trans conformation in the both proteins.
M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.
It has been showned that cationic alpha-helical peptides can be useful as nucleic acid-carrier molecules for gene transfer into cells. In order to investigate the significancemake sure of importance of the hydrophobic region in amphiphilic peptides in relation to their transfection ability, we have employed five kinds of peptides with a systematically varied hydrophobic-hydrophilic balance in the amphiphilic structures, and have evaluated the relationship between the structure and the gene transfer ability of the peptides into COS-7 cells. The peptides with a large hydrophobic region took alpha-helical structures, formed large aggregates and showed high transfection efficiency. Their high efficiency can be explained on the basis of their ability to form stable aggregates which can be internalized by endocytosis and remain resistant to digestion in lysosomal vesicles. Furthermore, it was suggested that the hydrophobic region of peptides plays an important role in the disruption of the endosomal membrane, which ca prevent the degradation of DNA in lysosomal vesicles. When peptides do not have so strong membrane-disruptive activity, but form aggregates which can be incorporated by endocytosis, the transfection efficiency can be recovered by the addition of an endosome-disruptive peptide.
Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.
11Z-9-Demethyl-9-benzyl- and 9-phenyl-retinals were synthesized stereoselectively from the beta-ionone analog-tricarbonyliron complexes and their interaction with bovine opsin was investigated.
Atrial, brain, and C-type natriuretic peptides of the atrial natriuretic peptide family are present in adrenal chromaffin cells, and are secreted with catecholamines by exocytosis. These peptides modulate the physiological functions of the cells such as synthesis and secretion of catecholamines in an autocrine manner interacting with natriuretic peptide receptors.
We examined the effects of riluzole, a neuroprotective drug, on voltage-dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced 22Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC50 of 5.3 microM. It was associated with a similar concentration-related reduction of veratridine-induced 45Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on 45Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced 22Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated 22Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced 45Ca influx. The inhibitory effect of riluzole on veratridine-induced 22Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced 22Na influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage-dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors.
BACKGROUND: Plasma atrial natriuretic peptide (ANP), mainly from the atrium, brain natriuretic peptide (BNP), mainly from the ventricle, norepinephrine (NE), and endothelin-1 (ET-1) levels are increased with the severity of congestive heart failure (CHF). Although a close correlation between the left ventricular end-diastolic pressure (LVEDP) and plasma ANP in patients with left ventricular dysfunction has been reported, it is not yet known which cardiac natriuretic peptide is a better predictor of high LVEDP in patients with CHF. METHODS: To investigate the biochemical predictors of the high LVEDP in patients with left ventricular dysfunction, we measured plasma ANP, BNP, NE, and ET-1 levels and the hemodynamic parameters in 72 patients with symptomatic left ventricular dysfunction. Stepwise multivariate regression analyses were also used to determine whether the plasma levels of ANP, BNP, NE, and ET-1 could predict high LVEDP. RESULTS: Although significant positive correlations were found among the plasma levels of ANP, BNP, ET-1, and NE and the LVEDP, only BNP (p = 0.0001) was an independent and significant predictor of high LVEDP in patients with CHF. In all eight patients with severe CHF measured for hemodynamics before and after the treatments, the plasma BNP levels decreased in association with the decrease of LVEDP, whereas other factors increased in some patients despite the decrease of LVEDP. CONCLUSIONS: These findings suggest that plasma BNP is superior to ANP as a predictor of high LVEDP in patients with symptomatic left ventricular dysfunction.
OBJECTIVE: Both the selective endothelin (ET) ETA receptor and mixed ETA/ETB receptor antagonists improve haemodynamics in patients and experimental models with congestive heart failure (CHF) and reduce the mortality in CHF rats. However, it remains unclear which of these antagonists is superior in the treatment of CHF. In addition, there is little information as to whether these ET receptor antagonists contribute to the neuroendocrine regulation and body fluid balance. We therefore investigated the cardiorenal and neurohumoral benefits of selective ETA receptor and mixed ETA/ETB receptor antagonists in CHF. METHODS: We administered acutely either the selective ETA receptor antagonist FR139317 (FR, n = 6, 1 and 10 mg/kg) or the mixed ETA/ETB receptor antagonist TAK-044 (TAK, n = 6, 1 and 3 mg/kg) to conscious dogs with CHF induced by rapid right ventricular pacing for ten days. RESULTS: Both FR and TAK decreased the cardiac pressures and the plasma atrial natriuretic peptide level and increased the cardiac output and urinary sodium excretion. FR increased the urine flow rate in association with an increased glomerular filtration rate and renal plasma flow, while TAK reduced the plasma aldosterone level. Neither antagonist increased the plasma renin activity or norepinephrine levels. CONCLUSIONS: These ETA/ETB antagonist. However, the long-term administration of a mixed ETA/ETB receptor antagonist would improve not only the haemodynamics but also prevent fluid retention by suppressing secretion of aldosterone during the treatment of chronic CHF.
OBJECTIVES: We 1) evaluated whether interleukin-6 (IL-6) is produced in the peripheral circulation in patients with congestive heart failure (CHF), 2) estimated the factors for increased IL-6, and 3) clarified the prognostic role of high plasma levels of IL-6 in patients with CHF. BACKGROUND: Although plasma levels of IL-6 have been reported to increase in patients with CHF, and production of IL-6 in endothelial cells and vascular smooth muscle cells has been postulated from in vitro studies, the origin of the increase of IL-6 in CHF remains unknown. Moreover, the prognostic value of a high plasma level of IL-6, independent of classic neurohumoral factors, remains to be elucidated. METHODS: A comparison was made of the plasma levels of IL-6 between the femoral artery and the femoral vein in 13 normal subjects and in 80 patients with CHF. In another study, we measured plasma IL-6 in 100 patients with CHF and follow-up data. RESULTS: Plasma IL-6 levels increased significantly from the femoral artery to the femoral vein in normal subjects and in patients with CHF. Arteriovenous IL-6 spillover in the leg increased with the severity of CHF. Among the hemodynamic variables and the various neurohumoral factors, the plasma norepinephrine (NE) level showed an independent and significant positive relation with the plasma IL-6 level in patients with CHF. Moreover, treatment with beta-adrenergic blocking agents showed an independent and significant negative relation with plasma IL-6 levels. In 100 patients, plasma IL-6 (p < 0.0001), NE (p = 0.0004) and left ventricular ejection fraction (0.015) were significant independent prognostic predictors by Cox proportional hazards analysis. CONCLUSIONS: Our findings indicate that the IL-6 spillover in the peripheral circulation increases with the severity of CHF and that the increase in plasma IL-6 is mainly associated with the activation of the sympathetic nervous system. High plasma levels of IL-6 can provide prognostic information in patients with CHF, independent of left ventricular ejection fraction and plasma NE, suggesting an important role for IL-6 in the pathophysiology of CHF.
Ribosomal modulation factor (RMF), a small basic protein, expresses transcriptionally in the stationary phase of Escherichia coli cells and binds to 50S ribosomal subunits. The RMF bound 70S ribosomes dimerize to form 100S particles that have no translational activity. In transferring the stationary cells to a fresh medium, the 100S particles release the RMF and dissociate to active 70S. The interconversion of ribosomes between active 70S and inactive 100S by RMF is a cellular mechanism controlling translation.
BACKGROUND: The current histological evaluation of the effects of endocrine therapy has difficulty in distinguishing pathologic degeneration caused by androgen ablation from residual poorly differentiated tumor. Therefore, we examined the changes in cell proliferation and apoptosis before and after endocrine therapy and analyzed whether they correlated with pathologic effects and histological differentiation. METHODS: Between January 1986 and December 1995, 52 patients with clinical stage B2 and C prostate cancer underwent radical prostatectomy after neoadjuvant endocrine therapy (median duration 3.8 months). Proliferative and apoptotic activities of pretreatment biopsy specimens and radical prostatectomy specimens were analyzed with MIB-1 monoclonal antibody and in situ end-labeling of fragmented DNA. RESULTS: The mean proliferative index (PI) of radical prostatectomy specimens was significantly lower than that of biopsy specimens (P = 0.000003) and the decrease in PI after endocrine therapy was significantly related to histological differentiation (P = 0.014). There was a weak relationship between the decrease in PI after endocrine therapy and pathologic effects (P = 0.054), while in pathologically effective cases (Grades 2 and 3), three out of 16 (19%) showed a < 50% decrease in PI after endocrine therapy, and may be regarded as having poorly differentiated tumors. The mean apoptotic index (AI) of prostatectomy specimens tended to be higher than that of biopsy specimens (P = 0.054). The increase in AI after endocrine therapy was not related to histological differentiation and pathologic effects. CONCLUSION: Pathologic effects caused by endocrine therapy may be in part misled by routine histopathologic staining and the change in PI may help in recognizing the pathologic effects of endocrine therapy and have adjunctive value for the interpretation of the effects of endocrine therapy.
Ribosomal proteins from a cyanobacterium Anabaena variabilis were analyzed by two-dimensional gel electrophoresis. We detected 21 protein spots of the small subunit and 29 protein spots of the large subunit. One of the spots was identified as L25 protein, which suggests that the reading frame sll1824 of Synechocystis is the L25 protein.
Endothelin (ET)-1 increases in plasma during congestive heart failure (CHF). Some ET antagonists improve hemodynamics, suggesting its potential benefits in the treatment of CHF. We examined the acute and chronic effects of a new ET receptor antagonist, T-0201 (Tanabe Seiyaku Co. Ltd., Japan), in CHF. To confirm the in vivo effects of T-0201, we observed the inhibitory effects of T-0201 (1-100 micrograms/kg) on the response of blood pressure to exogenously administered ET-1 (0.75 nmol/kg) in conscious normal dogs. Pretreatment with T-0201 significantly inhibited the ET-1-induced initial hypotension that is mediated by ETB receptors, and attenuated the subsequent hypertension, which is primarily mediated by ETA receptors. Thus, T-0201 at a dose of 100 micrograms/kg not only works as a potent ETA antagonist but also shows antagonist activity for ETB receptors in dogs. To evaluate the chronic therapeutic effects of T-0201, we administered T-0201 (0.3 mg/kg/day; n = 5) orally to dogs with CHF induced by rapid right ventricular pacing (22 days, 270 beats/min) for 15 days, beginning 8 days after pacing. T-0201 significantly prevented the deterioration of cardiorenal function during the development of CHF, expressed as a decrease in cardiac pressure and an increase in cardiac and urine output. These results suggest that chronic antagonism of both ET receptors prevents the progressive exacerbation of CHF.
A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.
pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in an Escherichia coli MC1061 transformant which grew on a plate containing 512 microg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.
The three zones of adrenal cortex are thought to arise from a single multipotential stem cell, but the mechanisms underlying the zonal differentiation during embryonic development of adrenal cortex are poorly understood. Employing subtraction cloning strategy, we isolated three distinct clones that were specifically expressed in the rat glomerulosa zone. One clone, named zona glomerulosa specific clone, encoded a membrane-spanning protein with a signal peptide at the N-terminus, six epidermal growth factor-like repeat motifs, and a transmembrane domain near the C-terminus. It was identified as a rat homolog of preadipocyte factor-1 (Pref-1), a factor involved in maintaining the undifferentiated status of preadipocyte. Immunohistochemical studies confirmed the presence of Pref-1 protein in the glomerulosa zone. Detailed examination revealed that the zone is divided into two layers; the first is a few-cells-thick layer present underneath the capsule (expressing both Pref-1 protein and aldosterone synthase cytochrome P450), and the second layer is beneath the first (containing Pref-1 protein but not aldosterone synthase). Moreover, another cell layer was found beneath the second layer and above the fasciculata zone, whose cells contained no Pref-1 protein, aldosterone synthase, or 11beta-hydroxylase. These findings suggest that a recently reported aldosterone synthase- and 11beta-hydroxylase-less cell layer between the two zones is composed of two kinds of cell: Pref-1 protein-positive and -negative cells. The level of Pref-1 message in the adrenal glands of animals having various pituitary-adrenal axis activities, as well as various plasma salt concentrations, correlated with the total number of glomerulosa cells. However, the specific content of Pref-1 message in a cell was fairly constant. When the adrenal gland was surgically enucleated and the remaining capsule regenerated, the level of Pref-1 transcript was significantly suppressed at the early phase. At this phase, only a minor population of the cortical cells expressed Pref-1 protein, most of these cells already expressing a fasciculata/reticularis-specific marker, inner zone antigen. These findings suggest that the capsular cells, mostly composed of the glomerulosa cells, may have potential for differentiating into other zones' cells, and the down-regulation of Pref-1 expression may be an important step in the adrenal zonal differentiation.
Early-stage heart failure (HF) is characterized by an increase in circulating atrial natriuretic peptide (ANP) without activation of the renin-angiotensin-aldosterone system (RAAS) or body fluid retention. To test the hypothesis that elevated endogenous ANP suppresses the RAAS, maintains body fluid balance, and regulates vascular tone in early-stage HF, we assessed the effects of short-term and long-term inhibition of ANP on cardiorenal and neurohormonal functions. Short-term antagonism was produced by bolus administration (3 mg/kg) of HS-142-1, an antagonist of guanylate-cyclase coupled ANP receptors, and long-term antagonism was produced by continuous infusion (1 mg/kg per h) of HS-142-1 for 8 h to dogs with early-stage HF induced by rapid ventricular pacing (270 beats/min, 8 days). In this experimentally produced HF, plasma ANP was significantly increased relative to the pre-pacing value, but not plasma renin activity (PRA) or plasma aldosterone level. HS-142-1 significantly suppressed plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) levels, markers of endogenous ANP activity, in both experiments. Although mean arterial pressure and cardiac output did not change significantly, pulmonary capillary wedge pressure and right atrial pressure were elevated in both experiments. While short-term inhibition of ANP did not change PRA and aldosterone levels, long-term inhibition significantly increased these hormonal levels, resulting in decreases in urine flow rate, urinary sodium excretion rate, glomerular filtration rate, and renal plasma flow. These findings suggest that endogenous ANP plays a critical role in regulating venovascular tone, inhibiting activation of RAAS, and maintaining renal functions in early-stage HF.