Effects of hydration on the electronic structure of an enzyme: implications for the catalytic function.
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Algorithms↗
Biomedical subjects
M Sakurai
Publications and source records attributed to M Sakurai.
Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.
The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.
Animals↗
Evidence for large structural fluctuations of the photobleached intermediate of photoactive yellow protein in solution.
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Journal Article↗
A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver.
Carbohydrates mediate their conversion to triglycerides in the liver by promoting both rapid posttranslational activation of rate-limiting glycolytic and lipogenic enzymes and transcriptional induction of the genes encoding many of these same enzymes. The mechanism by which elevated carbohydrate levels affect transcription of these genes remains unknown. Here we report the purification and identification of a transcription factor that recognizes the carbohydrate response element (ChRE) within the promoter of the L-type pyruvate kinase (LPK) gene. The DNA-binding activity of this ChRE-binding protein (ChREBP) in rat livers is specifically induced by a high carbohydrate diet. ChREBP's DNA-binding specificity in vitro precisely correlates with promoter activity in vivo. Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucose-responsive regulation. ChREBP is likely critical for the optimal long-term storage of excess carbohydrates as fats, and may contribute to the imbalance between nutrient utilization and storage characteristic of obesity.
Animals↗
Induction of phosphatidylinositol 3-kinase and serine-threonine kinase-like immunoreactivity in rabbit spinal cord after transient ischemia.
The mechanism of spinal cord injury has been thought to be related with tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. To evaluate the mechanism of such vulnerability of motor neurons, we attempted to make a reproducible model of rabbit spinal cord ischemia. Using this model, the inductions of phosphatidylinositol 3-kinase (PI3-k) and serine-threonine kinase (Akt) were investigated with immunohistochemical analyses for up to 7 days of the reperfusion following 15 min of ischemia in rabbit spinal cord. It has been demonstrated that both PI3-k and its downstream effector, Akt mediate growth factor-induced neuronal survival. Spinal cord sections from animals sacrificed at 8 h, 1, 2, and 7 days following the 15 min of ischemia were immunohistochemically evaluated using monoclonal antibodies for PI3-k and Akt. Following the 15 min of ischemia, the majority of the motor neurons showed selective cell death at 7 days of reperfusion. Immunoreactivity of PI3-k and Akt were induced at 8 h of reperfusion selectively in motor neuron cells. No glial cells and inter neurons were stained in the spinal cord sections. The activation of PI3-k and Akt protein at the early stage of reperfusion may be one of the factors responsible for the delay in neuronal death after spinal cord ischemia.
Animals↗
Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase.
A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.
3-Isopropylmalate Dehydrogenase↗
Role of the conserved Ser-Tyr-Lys triad of the SDR family in sepiapterin reductase.
Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser-Tyr-Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.
Alcohol Oxidoreductases↗
Effects of conditioned medium on activities of PAL, CHS, DAHP synthase (DS-Co and DS-Mn) and anthocyanin production in suspension cultures of Fragaria ananassa.
A conditioned medium (CM) prepared from cell suspension cultures of strawberry stimulated anthocyanin synthesis. The effect was significantly (P<0.05) greater than that of synthetic medium (SM), with macronutrient concentrations, carbohydrate concentrations and pH adjusted to those of CM. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) were monitored in the CM- and SM-cultured cells. PAL and CHS activities were found to increase significantly (P<0.05) in the CM-cultured cells. CHS transcript levels were higher in the CM-cultured cells compared to transcript abundance in SM-cultured cells. There was no significant difference in the DS-Mn and DS-Co activities of cells grown in conditioned or synthetic media.
Journal Article↗
Diphenylpyraline-responsive parkinsonism in cerebrotendinous xanthomatosis: long-term follow up of three patients.
A long-term follow-up study was made of three patients with cerebrotendinous xanthomatosis (CTX) associated with parkinsonism, two of whom were siblings. Besides typical CTX symptoms, all three patients showed severe parkinsonism. This observation has been rarely reported in CTX. The fact that the two siblings showed parkinsonism strongly suggests the genetic propensity to parkinsonism in these CTX patients. Positron emission tomography studies of the two patients revealed presynaptic dysfunction of the nigro-striatal dopaminergic system. Treatment with the reductase inhibitor hydroxymethyl glutaryl coenzyme successfully corrected the serum cholestanol level in the early stage of the disease, which, however, did not arrest the progression of clinical symptoms, particularly their parkinsonism. Clinically, levodopa had a little effect on parkinsonism, whereas an antihistamine drug, diphenylpyraline hydrochloride (DPP) had excellent effects on all three patients throughout the long-term follow up. The mechanism of the action of DPP on parkinsonism is unclear, however, the drug seems to be a therapeutic choice for treating parkinsonism in CTX.
Adult↗
Plasma levels of natriuretic peptides in relation to doxorubicin-induced cardiotoxicity and cardiac function in children with cancer.
BACKGROUND: Anthracyclines are effective anticancer drugs for childhood cancer with dose-limiting cardiotoxicity. Children who have received anthracyclines thus need periodical cardiac evaluation. The plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have been shown to increase in proportion to severity of cardiac dysfunction. We examined whether plasma levels of ANP and BNP, in addition to echocardiographic evaluation, can be used as specific markers for doxorubicin-induced cardiotoxic effects in children. PROCEDURE: Consecutively, 34 patients (18 boys and 16 girls) who had previously received doxorubicin-containing chemotherapy were enrolled in this study. Plasma ANP and BNP were assayed simultaneously at the time of first cardiac function evaluation by echocardiography. RESULTS: Of the 34 patients, 8 (23.5%) had left ventricular dysfunction as assessed by echocardiography. Both ANP and BNP plasma levels in these patients were significantly elevated in comparison with healthy controls (P < 0.01) or patients with normal cardiac function (P < 0.05). It should be also noted that ANP and BNP levels were correlated significantly with cardiac systolic function, but not with diastolic function. CONCLUSIONS: These results suggest that plasma ANP and BNP levels could be markers for doxorubicin-induced cardiotoxicity in children. Measurement of natriuretic peptide levels during treatment may allow earlier-identification of individuals at risk for severe cardiac damage.
Adolescent↗
Thoracodorsal artery as a free arterial graft for myocardial revascularization.
When complete arterial revascularization of coronary circulation cannot be accomplished using the internal thoracic artery and other conventionally used arterial conduits, the thoracodorsal artery may be an excellent alternative. We report the use of the thoracodorsal artery as a free arterial graft in 3 patients, describe the harvesting technique, and review the anatomy.
Aged↗
Cleft on tricuspid anterior leaflet.
We report a case of isolated congenital tricuspid regurgitation caused by a cleft in the anterior tricuspid leaflet associated with a patent foramen ovale. Preoperative echocardiography revealed severe tricuspid regurgitation resulting from anterior tricuspid leaflet prolapse. The patient underwent successful tricuspid valve repair with simple cleft suture and annuloplasty and direct closure of the patent foramen ovale.
Cardiac Surgical Procedures↗
Dihydroxynortropane alkaloids from calystegine-producing plants.
Three dihydroxynortropanes, 2alpha,7beta-dihydroxynortropane, 2alpha,3beta-dihydroxynortropane, and 3alpha,7beta-dihydroxynortropane, were isolated from calystegine-producing plants in the families Convolvulaceae and Solanaceae. 2alpha,7beta-Dihydroxynortropane was isolated from six species in the Convolvulaceae whereas only Calystegia soldanella contained it and 2alpha,3beta-dihydroxynortropane. Although neither of these were detectable in three species tested in the Solanaceae, 3alpha,7beta-dihydroxynortropane was, however, isolated from Duboisia leichhardtii.
Alkaloids↗
Significance of reduced uptake of iodinated fatty acid analogue for the evaluation of patients with acute chest pain.
OBJECTIVES: To assess whether 15-(p-[iodine-123] iodophenyl)-3-(R,S) methylpentadecanoic acid (BMIPP) imaging can identify previous ischemic areas, BMIPP SPECT was performed in patients with acute chest pain to compare its findings with those of technetium-99m-tetrofosmin (tetrofosmin) SPECT and coronary angiography. BACKGROUND: Basic studies indicate that BMIPP can identify previous ischemia as reduced tracer uptake. METHODS: This study prospectively enrolled 111 consecutive patients with acute chest pain without myocardial infarction. Tetrofosmin SPECT was performed at rest within 24 h after the last episode of chest pain. Coronary angiography and BMIPP SPECT were also performed on the following day. RESULTS: Sixty-four of the 87 patients with coronary stenosis or spasm showed BMIPP abnormalities corresponding to the areas of coronary abnormalities (sensitivity 74%), whereas only 33 of them showed perfusion abnormalities (sensitivity 38%) (p < 0.001). Of 24 patients [corrected] without coronary stenosis or spasm, 22 showed normal BMIPP SPECT (specificity = 92%) [corrected] and 23 showed normal tetrofosmin SPECT (sensitivity = 96%) [corrected]. Coronary stenosis was more often seen in the group with abnormal tetrofosmin/abnormal BMIPP (82%) and with normal tetrofosmin/abnormal BMIPP (69%) than in the group with normal tetrofosmin/normal BMIPP (36%) (p < 0.05). Coronary spasm was observed more often in the group with abnormal tetrofosmin/abnormal BMIPP (83%) and with normal tetrofosmin/abnormal BMIPP (90%) than in the group with normal tetrofosmin/normal BMIPP (27%) (p < 0.05). The extent and severity scores of tetrofosmin and BMIPP in the patients with organic stenosis were significantly higher than those of patients with no organic stenosis or spasm (p < 0.0001). CONCLUSIONS: These data indicate that BMIPP SPECT may specifically identify previous ischemic lesions due to coronary stenosis or spasm in patients with acute chest pain.
Aged↗
Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapenems Part IV. 2-Alkyl substituents containing cationic heteroaromatics linked via a C-C bond.
The synthesis and biological activity of a novel series of 2-alkyl-4-pyrrolidinylthio-beta-methylcarbapenems containing a variety of cationic heteroaromatic substituents linked via a C-C bond is described. As a result of these studies, we selected FR21818 (In) as a candidate compound for development. FR21818 exhibited a well balanced spectrum of antibacterial activity, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), excellent urinary recovery, good stability against renal dehydropeptidase-I (DHP-I). no antigenicity and mutagenicity, weak toxicities, and good efficacy and therapeutic effect on mice systemic infections. Affinities to PBP's, permeability of outer membrane, and plasma levels in mice, dog, and cynomolgous monkey of FR21818 are also reported.
Animals↗
Choroidal circulation in diabetic patients.
PURPOSE: To evaluate choroidal circulatory changes in diabetic patients with and without background diabetic retinopathy (BDR) by measuring the retrobulbar circulation with colour Doppler imaging (CDI). METHODS: CDI was used to measure circulatory parameters of the PCA (posterior ciliary artery), CRA (central retinal artery), OA (ophthalmic artery) and the respective veins in 73 diabetic patients and 22 controls in a sitting posture. Among the diabetic patients, 38 patients were without diabetic retinopathy (NDR) and 35 had BDR. A non-parametric Kruskal-Wallis test with a Dunn correction was used for data analysis. RESULTS: End-diastolic velocity (EDV) in the PCA was decreased (2.55+/-0.80 cm/s) and resistivity index (RI) in the PCA was increased (0.70+/-0.08) in BDR patients compared with the control patients' EDV (3.23+/-1.08 cm/s, p = 0.01) and RI (0.62+/-0.06, p = 0.0003). RI in the CRA was significantly higher in the BDR group (0.74+/-0.09) than in the control group (0.68+/-0.08, p = 0.006). RI in the OA was significantly higher in the BDR group (0.87+/-0.06) compared both with the NDR group (0.83+/-0.07) and with the control group (0.81+/-0.06; p = 0.007, p = 0.004). NDR patients had a significantly higher RI in the PCA (0.67+/-0.08) than control patients (0.62+/-0.06, p = 0.01, while the CRA RI (0.71+/-0.09) did not show significant differences from the control group (0.69+/-0.08, p = 0.32). Decreased EDV in the CRA was detected in NDR patients (2.16+/-0.76 cm/s) compared with the controls (2.72+/-0.92 cm/s, p = 0.007). CONCLUSION: The results from this study suggest that not only the retinal but also the choroidal circulation is affected in NDR and BDR patients.
Analysis of Variance↗
Differential induction of LTP and LTD is not determined solely by instantaneous calcium concentration: an essential involvement of a temporal factor.
Two opposite types of synaptic plasticity in the CA1 hippocampus, long-term potentiation (LTP) and long-term depression (LTD), require postsynaptic Ca2+ elevation. To explain these apparently contradictory phenomena, the current view assumes that a moderate postsynaptic increase in Ca2+ leads to LTD, whereas a large increase leads to LTP. No detailed study has so far been attempted to investigate whether the instantaneous Ca2+ elevation level differentially induces LTP or LTD. We therefore used low-frequency (1 Hz) stimulation of Schaffer collateral/commissural fibers in rat hippocampal slices, during a Mg2+-free period, as the conditioning stimulus to investigate this. This allowed low-frequency afferent stimulation to cause a postsynaptic Ca2+ influx because the voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor-channels by Mg2+ was removed. When delivered during the Mg2+-free period, a single pulse, as well as 2-600 pulses, induced LTP that was occluded with tetanus-induced LTP. To decrease the Ca2+ influx, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were completely blocked by the addition of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the conditioning medium, in which 1 Hz afferent stimuli (1-600 pulses) induced less LTP and never induced LTD. To further reduce the Ca2+ influx, NMDA receptors were partially blocked with D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5). A small number of 1 Hz stimuli, however, never induced LTD. Only when the conditioning stimuli exceeded 200 pulses was LTD induced. The present findings provide definitive evidence that protracted conditioning is a prerequisite for the induction of LTD. Thus, not only the amplitude but also the duration of postsynaptic Ca2+ elevation could be essential factors for differentially inducing LTP or LTD.
2-Amino-5-phosphonovalerate↗
Differential responsiveness of cord and adult blood monocytes to hepatocyte growth factor.
Monocytes as antigen-presenting cells play an important role in host defence. There are several cytokines affecting monocyte function. We demonstrate that both adult and cord blood monocytes constitutively express hepatocyte growth factor (HGF) receptor, MET. HGF significantly down-regulated MET expression of adult blood monocytes, compared with cord blood monocytes, when cultured either in RPMI-1640 containing 10% FBS or serum-free medium. Surface levels of MET correlated with c-met mRNA levels both in adult and cord blood when cultured. MET expression was down-regulated by treating with actinomycin D or cycloheximide. HGF stimulated DNA synthesis of adult monocytes, but not cord blood. HGF enhanced antigen-presenting capacity of adult blood monocytes but not cord blood monocytes. HGF up-regulated HLA class I expression in adult monocytes but not in cord blood monocytes. The current results suggest that the failure of cord blood monocytes to respond to HGF may be responsible, in large part, for their functional immaturity.
Adult↗