Proton inventory of the genomic HDV ribozyme in Mg(2+)-containing solutions.
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Cations, Divalent↗
Biomedical subjects
S Nakano
Publications and source records attributed to S Nakano.
Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism.
Hepatitis delta virus (HDV) uses genomic and antigenomic ribozymes in its replication cycle. We examined ribozyme self-cleavage over eight orders of magnitude of Mg(2+) concentration, from approximately 10(-9) to 10(-1) M. These experiments were carried out in 1 M NaCl to aid folding of the ribozyme and to control the ionic strength. The concentration of free Mg(2+) ions was established using an EDTA-Mg(2+) buffered system. Over the pH range of 5-9, the rate was independent of Mg(2+) concentration up to 10(-7) M, and of the addition of a large excess of EDTA. This suggests that in the presence of 1 M NaCl, the ribozyme can fold and cleave without using divalent metal ions. Brønsted analysis under these reaction conditions suggests that solvent and hydroxide ions may play important roles as general base and specific base catalysts. The observed rate constant displayed a log-linear dependence on intermediate Mg(2+) concentration from approximately 10(-7) to 10(-4) M. These data combined with the shape of the pH profile under these conditions are consistent with the binding of at least one structural divalent metal ion that does not participate in catalysis and binds tighter at lower pH. No evidence for a catalytic role for Mg(2+) was found at low or intermediate Mg(2+) concentrations. Addition of Mg(2+) to physiological and higher concentrations, from 10(-3) to 10(-1) M, revealed a second saturable divalent metal ion which binds tighter at high pH. The shape of the pH profile is inverted relative to that at low Mg(2+) concentrations, consistent with a general acid-base catalysis mechanism in which a cytosine (C75) acts as the general acid and a hydroxide ion from the divalent metal ion, or possibly from solvent, acts as the base. Overall, the data support a model in which the HDV ribozyme can self-cleave by multiple divalent ion-independent and -dependent channels, and in which the contribution of Mg(2+) to catalysis is modest at approximately 25-fold. Surface electrostatic potential maps were calculated on the self-cleaved form of the ribozyme using the nonlinear Poisson-Boltzmann equation. These calculations revealed several patches of high negative potential, one of which is present in a cleft near N4 of C75. These calculations suggest that distinct catalytic and structural metal ion sites exist on the ribozyme, and that the negative potential at the active site may help shift the pK(a) for N3 of C75 toward neutrality.
Catalysis↗
Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.
Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.
Base Composition↗
Effects of cilnidipine on nitric oxide and endothelin-1 expression and extracellular signal-regulated kinase in hypertensive rats.
We evaluated the effects of cilnidipine, a long-acting Ca(2+) channel antagonist, on endothelial nitric oxide synthase (eNOS), preproendothelin-1 and endothelin ETA receptor expression in the left ventricle, and evaluated the relations between these effects and coronary microvascular remodeling and extracellular signal-regulated kinases belonging to one subfamily of mitogen-activated protein kinases in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Cilnidipine (DOCA-cilnidipine, 1 mg/kg/day, subdepressor dose) or vehicle (DOCA-vehicle) was given after induction of DOCA-salt hypertension for 5 weeks. The eNOS mRNA and protein expression in the left ventricle was significantly lower in DOCA-vehicle than in control rats and significantly higher in DOCA-cilnidipine than in DOCA-vehicle rats. Preproendothelin-1 and endothelin ETA receptor expression levels and phospho-p42/p44 extracellular signal-regulated kinase activities were significantly increased in DOCA-vehicle compared with control rats and significantly suppressed in DOCA-cilnidipine compared with DOCA-vehicle rats. DOCA-vehicle rats showed a significant increase in the wall-to-lumen ratio, perivascular fibrosis and myocardial fibrosis, with all these parameters being significantly improved by cilnidipine. These results led us to conclude that phospho-p42/p44 extracellular signal-regulated kinase activities may contribute to the coronary microvascular remodeling of DOCA rats and that protective effects of cilnidipine on cardiovascular remodeling may be at least in part mediated by an increased eNOS expression and a decreased endothelin-1 and endothelin ETA receptor expression in the left ventricle.
Animals↗
[Renovascular hypertension coexisting with an aldosteronoma].
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Adenocarcinoma↗
Impact of diabetes mellitus on the prognosis of patients with hepatocellular carcinoma.
BACKGROUND: The majority of patients with hepatocellular carcinoma (HCC) have coexisting cirrhosis or chronic hepatitis, often complicated by diabetes mellitus. In the current study, the authors evaluated the impact of diabetes mellitus on the prognosis of patients with HCC. METHODS: Among 581 patients with HCC who had been diagnosed and treated between 1990 and 1999, survival was compared between those patients with and those patients without diabetes mellitus. The rate of disease recurrence after treatment also was analyzed. RESULTS: Ninety-two patients (15.8%) had diabetes mellitus. There was no significant difference with regard to patient characteristics (i.e., age, gender, or alcohol intake) or liver function between those patients with and those patients without diabetes mellitus. No differences were observed in survival between patients with diabetes mellitus and patients without it. Among the 195 patients with a solitary HCC lesion measuring < or = 3 cm in greatest dimension, the survival of the 32 patients with diabetes mellitus was significantly poorer than that of the 163 patients without diabetes mellitus (P = 0.0273), despite no apparent difference in liver function between the 2 groups. On multivariate analysis, diabetes mellitus was found to be an independent factor predicting lower survival after treatment (P = 0.0077) among patients with a solitary HCC lesion measuring < or = 3 cm in greatest dimension. No difference in the rate of recurrence was observed between the two groups in all the patients and in those patients with a solitary HCC lesion measuring < or = 3 cm in greatest dimension. CONCLUSION: The results of the current study indicated that the presence of diabetes mellitus worsens the prognosis of patients with a solitary HCC lesion measuring < or = 3 cm in greatest dimension; it appears to impact prognosis in patients with HCC when HCC is treatable, based on the size and the number of lesions. However, diabetes mellitus did not appear to affect the prognosis in the general population of patients with HCC. Based on the current study data, diabetes mellitus does not appear to modify the progression of HCC and its recurrence after treatment, but it does appear to worsen the prognosis of patients with HCC by means of a rapid decline in remnant liver function caused by repeated treatment of HCC.
Aged↗
Mass mortality of the Japanese pearl oyster Pinctada fucata martensii in relation to water temperature, chlorophyll a and phytoplankton composition.
Mass mortalities of the Japanese pearl oyster Pinctada fucata martensii have widely occurred in western Japan since 1994. The causes of these mass mortalities are at present not thoroughly understood. In this study, we investigated oyster survival in relation to some environmental factors such as water temperature, concentration of chlorophyll a and density or composition of phytoplankton. The examined mass mortality occurred from September to December 1998, and the color on the adductor muscle of the oysters was red-brown, suggesting an infectious disease. Oysters that became moribund during the experiment lost weight, while the weight of unaffected oysters increased. The cell density of Nitzschia spp., an inedible algae for the oyster, in Uchiumi Bay increased before and during the mass mortality event. From the results of our study, we hypothesize that P. fucata martensii was weakened by starvation because of the dominance of inedible food and then contracted an infectious disease that resulted in mortality.
Animals↗
Inclusion body myositis: expression of extracellular signal-regulated kinase and its substrate.
OBJECTIVE: To assess abnormal intracellular signal transduction in inclusion body myositis (IBM). BACKGROUND: Mitogen-activated protein kinases (MAPKs) play pivotal roles in intracellular signal transduction and regulate cell growth and differentiation. Upon their activation, MAPKs translocate from the cytoplasm into the nucleus. DESIGN/METHODS: The authors investigated the localization of several forms of the MAPK family-extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 MAPK (p38)-in 10 patients with sporadic IBM and in 52 control subjects. The relationship between the localization of immunopositive deposits and nuclei was tested with bis-benzimide. RESULTS: Vacuolated fibers in IBM displayed very strong focal immunoreactivity of ERK, but not of JNK or p38. The ERK-positive deposits in these vacuolated fibers colocalized with the nuclear substrate of ERK, Elk-1. ERK- and Elk-1-positive deposits were located frequently on the surface of the nuclei in vacuolated fibers in IBM. Similar findings to those of sporadic IBM were observed in three patients with distal myopathy with rimmed vacuoles, but not in eight normal or the other 41 disease controls. CONCLUSION: There is evidence for impaired molecular transport to the nucleus from the cytoplasm in the vacuolated fibers in IBM. This could be due to cytoplasmic aggregation of ERK and Elk-1 or to abnormal nuclear pore machinery involved in the transport of ERK and its substrate upon ERK activation.
Adult↗
Reciprocal subsidies: dynamic interdependence between terrestrial and aquatic food webs.
Mutual trophic interactions between contiguous habitats have remained poorly understood despite their potential significance for community maintenance in ecological landscapes. In a deciduous forest and stream ecotone, aquatic insect emergence peaked around spring, when terrestrial invertebrate biomass was low. In contrast, terrestrial invertebrate input to the stream occurred primarily during summer, when aquatic invertebrate biomass was nearly at its lowest. Such reciprocal, across-habitat prey flux alternately subsidized both forest birds and stream fishes, accounting for 25.6% and 44.0% of the annual total energy budget of the bird and fish assemblages, respectively. Seasonal contrasts between allochthonous prey supply and in situ prey biomass determine the importance of reciprocal subsidies.
Animals↗
The role of neoadjuvant radiochemotherapy using low-dose fraction cisplatin and 5-fluorouracil in patients with carcinoma of the esophagus.
OBJECTIVE: We clarified the role of neoadjuvant radiochemotherapy in patients with carcinoma of the esophagus and compared it to neoadjuvant chemotherapy. METHODS: We retrospectively examined 40 patients diagnosed with advanced thoracic esophageal carcinoma who underwent neoadjuvant therapy followed by esophagectomy between 1993 and 1999. We divided them into 2 groups: radiochemotherapy (17) and chemotherapy (23). Radiochemotherapy patients underwent 40 Gy radiation and low-dose fraction cisplatin (7 mg/body/day, 5 days a week x 4 weeks) and 5-fluorouracil (350 mg/body/day x 28 days). Chemotherapy patients received high-dose fraction cisplatin/5-fluorouracil involving 2 courses of cisplatin (70 mg/m2/day on day 1) and 5-fluorouracil (700 mg/m2/day on days 1-5). RESULTS: Complete pathological response was 17.6% in the radiochemotherapy group and 0% in the chemotherapy group respectively. No hospital mortality occurred in the radiochemotherapy group, and 1 of the 23 chemotherapy patients died in the hospital due to postoperative complications. The incidence of residual tumors was significantly higher in the chemotherapy group (34.8%) than in the radiochemotherapy group (0%). Actuarial survival in the radiochemotherapy group at 1 year was 80.2% and at 3 years 53.5%. Actuarial survival in the chemotherapy group at 1 year was 56.5% and at 3 years 30.4%. CONCLUSIONS: Histological effectiveness was greater in patients treated with preoperative radiochemotherapy than those treated with preoperative chemotherapy. The combination of radiation and low-dose fraction CDDP/5-FU thus is first choice in neoadjuvant radiochemotherapy for the advanced esophageal carcinoma.
Aged↗
P39 immunoreactivity in glial cytoplasmic inclusions in brains with multiple system atrophy.
Glial cytoplasmic inclusions (GCIs) characteristically occur in the oligodendrocytes of patients with multiple system atrophy (MSA). Cyclin-dependent kinase 5 (Cdk5) regulates cytoskeletal dynamism through phosphorylation of cytoskeletal proteins such as neurofilament proteins, tau and microtubule-associated protein 2. We examined the immunohistochemical localization of p39, a Cdk5 activator, in human brain specimens obtained post mortem from controls and patients with MSA. Among control specimens, p39 immunoreactivity was found in some neuronal somata and axons. Similar immunoreactivity was detected in MSA neuronal cell bodies and axons, but a number of inclusions were heavily labeled. These results suggest that p39 and Cdk5 are up-regulated in oligodendrocytes and may be involved in the formation of GCIs through phosphorylation of cytoskeletal proteins in oligodendrocytes in MSA.
Aged↗
Changes in arterial and portal perfusion in embolized and nonembolized hepatic lobes after portal vein embolization evaluated by helical computed tomography.
We evaluated the changes in hepatic arterial and portal perfusion in nonembolized as well as in embolized lobes after portal venous branch embolization (PVE) with dynamic helical computed tomography (CT). Six patients with hepatic malignancies, who underwent PVE prior to a subsequent hepatectomy, were the subjects of this study. We performed CT examinations before PVE and 2 weeks after PVE to make a volumetric analysis. At the same time, we performed single-location dynamic sequences after the injection of a 50-ml bolus of contrast medium, and we then created time-density curves from circular regions of interest drawn over the aorta, parenchyma of the right and left lobe of the liver, and spleen. We calculated the arterial perfusion index (ml/min per ml of tissue) and the portal perfusion index by dividing the maximum rate of enhancement of the liver before and after the splenic peak by the peak aortic enhancement. We then calculated the arterial and portal flows (ml/min) from the perfusion index and values of CT volumetry. In the right lobe, where the portal flow was occluded, the arterial perfusion index and flow increased significantly after PVE. In contrast, the arterial perfusion index and flow both decreased in the left lobe after PVE in a reverse response to the increase in the portal perfusion index and flow. The total arterial flow of the liver thus seemed to slightly increase; however, the change was not significant. By performing PVE an increased arterial perfusion was induced in the embolized lobe, with a concomitant decrease in arterial perfusion in the nonembolized lobe.
Aged↗
Serial changes in signal intensities of the adjacent discs on T2-weighted sagittal images after surgical treatment of cervical spondylosis: anterior interbody fusion versus expansive laminoplasty.
BACKGROUND: There have been many reports about newly developed degenerative changes in the adjacent segments after anterior interbody fusion. It is a controversial issue whether the adjacent-segment disease in patients treated by anterior interbody fusion is the result of progressive cervical spondylosis at the adjacent levels or is caused by the arthrodesis. The aim of this study is to clarify the difference in postoperative effect on the adjacent segments between anterior interbody fusion and expansive laminoplasty. METHOD: This study included 14 patients who underwent pre- and postoperative MR images at 6 and 12 months. Seven patients underwent cervical interbody fusion and the other 7 patients underwent expansive laminoplasty. Disc degeneration was evaluated semiquantitatively by calculating the degenerative index (DI) that is a ratio of the intensity in the disc to that in the upper cervical cord. FINDINGS: In the anterior interbody fusion group, the adjacent disc intensities decreased within 12 months (F = 20.42; P < 0.01). The pre-operative mean DI was 0.59 +/- 0.16. The post-operative mean DIs were 0.56 +/- 0.16 at 6 months and 0.47 +/- 0.16 at 12 months. In the expansive laminoplasty group, the signal intensities of both the adjacent discs and the discs within the range of laminoplasty had no serial changes during the same period (F = 2.67; P = 0.09 and F = 0.15; P = 0.87 respectively). INTERPRETATION: Anterior interbody fusion had a significant influence on the adjacent discs even as soon as 12 months after surgery, but laminoplasty had no influence on them during the same period.
Adult↗
TCV-116 stimulates eNOS and caveolin-1 expression and improves coronary microvascular remodeling in normotensive and angiotensin II-induced hypertensive rats.
Angiotensin II (Ang II) plays an important role as a modulator of vascular structure and function in arterial hypertension. This study investigated the effects of an Ang II type 1 receptor antagonist, TCV-116, on endothelial nitric oxide synthase (eNOS) mRNA and protein expression, and NOS activity and eNOS regulatory protein caveolin-1 protein expression in the left ventricle of Wistar-Kyoto rats treated for 2 weeks with Ang II (200 ng/kg/min) and evaluated these relations to myocardial remodeling. Rats given Ang II alone (ANGII) were compared with rats also receiving TCV-116 (ANGII-TCV). The eNOS mRNA and protein levels, and NOS activity and caveolin-1 protein expression in the left ventricle were significantly decreased in ANGII compared with control rats (CON), and were significantly increased in ANGII-TCV compared with ANGII. Moreover, compared with CON, the eNOS and caveolin-1 expression was significantly greater in CON treated with TCV-116. ANGII showed a significant increase of the wall-to-lumen ratio, perivascular and myocardial fibrosis, and type I collagen mRNA expression, with all these parameters being significantly improved by TCV-116. Thus, coronary microvascular and myocardial remodeling in normotensive and Ang II-induced hypertensive rats was significantly ameliorated by a subdepressor dose of TCV-116, which may be at least in part mediated by an increase in local eNOS mRNA and protein expression, and NOS activity in the left ventricle.
Angiotensin II↗
Novel cell lines established from a human myxoid malignant fibrous histiocytoma arising in the uterus.
Two cell lines (Nara-H and Nara-F) with different phenotypes were established from a myxoid MFH of the uterus. In vitro, Nara-F grew in sheets showing a storiform arrangement and Nara-H in raised colonies. Although tumors generated in nude mice shared similar morphological features of abundant myxoid tumor in Nara-H and -F, the pleomorphic component was conspicuous in Nara-F. Both cell lines produced hyaluronic-acid but CD44 was expressed only in Nara-H. Estrogen receptor alpha (ER alpha) and progesterone receptor (PgR) were detected in Nara-H. Nara-F was positive for ER beta and PgR. Among hormonal agents, the response to the anti-estrogen tamoxifen was more sensitive than progesterone agents. This report illustrates the characteristics of these newly established cell lines, and presents the possibility of an adjuvant hormonal therapy for MFH.
Aged↗
Long-term enhancement of synaptic transmission induced by veratridine in rat CA3 hippocampal neurons.
Veratridine is a neurotoxin that induces persistent activation of sodium channels in excitable cells. We investigated the effects of this toxin on excitatory synaptic transmission in CA3 neurons of juvenile rat hippocampus using whole-cell patch-clamp and field-potential recordings. The population spikes evoked by electrical stimulation of the mossy fiber were gradually enhanced after washout of veratridine (0.3 microM), but they were not enhanced by the co-application of veratridine and an N-methyl-D-aspartate (NMDA) receptor antagonist (D-APV, 30 microM). When a pipette solution contained QX-314 that antagonized the effect of veratridine in the recorded neuron, oscillatory membrane depolarization appeared in the early stage during bath-application of veratridine and gradually decreased in the late stage. After washout of veratridine, however, the oscillatory depolarization was gradually restored and maintained for at least 3 h. This oscillatory depolarization was also abolished by D-APV. We suggest that the activation of NMDA receptors is involved in the veratridine-induced long-lasting enhancement in the excitatory synaptic transmission in rat CA3 hippocampal neurons.
2-Amino-5-phosphonovalerate↗
Assessment of styrene oligomers eluted from polystyrene-made food containers for estrogenic effects in in vitro assays.
Recently, several substances from among the huge numbers of chemicals used by mankind have been implicated as instigators of disrupted endocrine function and related human health problems. Polystyrene (PS) is one of the most frequently used resins in the world, and the styrene oligomer dissolved out from PS has been designated as a potential trigger of estrogen-like activity in the Wingspread Declaration and the Japan Environment Agency's SPEED98 [JEA (Japan Environment Agency) Strategic Problem on Environmental Endocrine Disruptors '98 (SPEED) '98), http://www.env.go.jp/en/pol/speed98/sp98.html]. In order to assess the endocrine disrupting effect of styrene oligomers, we tested one styrene monomer (SM), three styrene dimers (SDs) and seven styrene trimers (STs), newly isolated from optical isomers, known to dissolve in small amounts from cup noodle containers made of polystyrene by the estrogen receptor binding assay, luciferase reporter gene assay, and human breast cancer cell MCF-7 proliferation assay. In all three tests, none of the SM, SDs and STs showed any significant activity. Accordingly, we concluded that these substances have no estrogenic activity.
Animals↗
Immunohistochemical analysis of distribution of RON receptor tyrosine kinase in human digestive organs.
The immunohistochemical distribution of RON receptor tyrosine kinase in digestive organs of both human fetus and adult, including the esophagus, stomach, duodenum, small intestine, colon, rectum, liver, gallbladder, pancreas, and spleen, was investigated semiquantitively using an affinity-purified rabbit polyclonal antibody. RON was observed to be widely distributed throughout various digestive organs and cell types in humans. The immunoreactivity for RON was observed in the epithelium of the esophagus, small intestine, colon, hepatocytes, Kupffer cells, and splenic macrophages both in the adult and the fetus, suggesting that the MSP/RON signaling pathway possesses the proper biological properties to possibly be involved in morphogenesis or differentiation of cells in these organs and cell types. Several organs differed in immunoreactivity between adult and fetus. No immunoreactive cells were found in the pancreas of adults; however, immunoreactivity was observed in acinar cells and in some of the duct or ductular cells and endocrine cells of the islet of the fetus. Similarly, immunoreactivity was not observed in gastric mucosa except in the intestinal metaplastic cells in adults; however, immunoreactivity was found in the foveolar epithelium of the stomach of the fetus. Although the biological significance of RON in malignancy is unclear, the presence of RON immunoreactivity in the fetus and it lack in the adult may indicate that RON is a oncofetal substance in human pancreas and stomach.
Adult↗