Spontaneous proliferation of peripheral blood mononuclear cells as an indicator for insufficient plasma levels of FK 506 in the early phase after liver transplantation.
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Animals↗
Biomedical subjects
S Kanaya
Publications and source records attributed to S Kanaya.
[Therapeutic effectiveness of 131I-MIBG on malignant pheochromocytoma--results of long-term follow-up].
The therapeutic response of 131I-MIBG was evaluated in 4 patients with malignant pheochromocytoma who had been treated with 131I-MIBG and followed-up over 5 years. The patients were 2 men and 2 women with ages ranging from 41 to 69 years old (mean 53 years). The primary tumors in 3 of 4 patients had been resected four to eight years before 131I-MIBG treatment. One patient was diagnosed as adrenal pheochromocytoma, and two were retroperitoneal paraganglioma. And in one patient, the resection of primary mediastinal tumor was not performed due to the adhesion to pericardium but the diagnosis of paraganglioma was obtained by biopsy of bone lesion. All patients showed the clear accumulation of 131I-MIBG in tumor on scintigraphy. The number of doses of 131I-MIBG ranged from one to three times with 3.7 GBq per administration and a cumulative activity from 3.7 to 11.1 GBq. Treatment effect was obvious in one patient with lung, bone, and lymph node metastases whose cumulated absorbed dose with 11.1 GBq of 131I-MIBG exceeded over 150 Gy. At the present time, the duration of survival since the beginning of initial 131I-MIBG therapy is over 5 yrs. The other three patients, however, showed little effects, and died with the disease in 2.6 to 4.1 years after the initial 131I-MIBG therapy. 131I-MIBG will become a promising agent for therapy in patients with malignant pheochromocytoma with high degree of accumulation.
3-Iodobenzylguanidine↗
[The nature of the external ionic modulation of the myocardial electrical propagation: importance of the safety factor].
The mechanisms of conduction change depending on the extracellular K+ and Ca2+ concentrations ([K+]o and [Ca2+]o, respectively) were investigated. Simultaneous measurements of active and passive membrane properties and net membrane excitability were fulfilled by arranging the intra- and extracellular microelectrodes in a superfused and paced guinea pig papillary muscle. Internal longitudinal resistance (ri), as a parameter of passive property, was evaluated by the intra- and extracellular voltage ratio. The maximum upstroke rate (Vmax) was used as an active property. Apparent threshold potential (Vth) was defined by the breakpoint in the action potential upstroke fitted semilogarithmically. Graded rise in [K+]o (from 2.7 to 15.0 mM) evoked a progressive fall in Vmax, and was associated with less negative resting membrane potential and constant ri. Conduction velocity (theta) was the maximum in 9.0 mM [K+]o ("supernormal" conduction) but not in 2.7 mM [K+]o which gave the greatest Vmax ("subnormal" conduction). Safety factor of conduction (S), as an index of net excitability, could most readily account for the [K+]o-dependent change in theta. This was true also in the case of [Ca2+]o elevation (from 0.9 to 5.4 mM). These results indicate that the cation-modulated propagation is governed by the cable theory including S as a matrix of combined active and passive properties.
Action Potentials↗
Spontaneous proliferation of peripheral blood lymphocytes as an indicator of intragraft immune activation in liver transplant patients.
The presence of activated lymphocytes in the blood of transplant recipients is considered to be a marker of an ongoing immune process. In this study, the clinical utility of spontaneous proliferation (SP) of peripheral blood mononuclear cells (PBMC) as a marker of in vivo activation was evaluated by in vitro [3H]thymidine incorporation in 22 patients in the 1st month after liver transplantation. Also immune activation in the graft was monitored by transplant aspiration cytology (TAC-A). In the study period, there were only 2 mild episodes of clinical acute rejection, where SP of PBMC was 1290 and 1541 cpm (vs. 99 cpm averaged in healthy controls). Though SP of PBMC was also increased in systemic infections, a significant positive correlation was observed between SP of PBMC and TAC-A score. In all episodes where TAC-A score was elevated to above 2, SP of PBMC was simultaneously increased to more than 1000 cpm. In 8 patients prophylactically treated with the anti-interleukin-2-receptor antibody, BT563, SP of PBMC was significantly lower compared to 7 patients treated with antithymocyte globulin. It is suggested that SP can be a reliable parameter of the in vivo activation of lymphocytes, which accompanies immune activation in liver graft, and is potentially useful as a sensitive, although rather non-specific, and non-invasive monitoring of intragraft alloresponse.
Adult↗
[Quantitative analysis of 123I-metaiodobenzylguanidine myocardial scintigraphy by myocardial uptake using a phantom].
To evaluate the quantitative analysis of 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy, total injected dose measured by first pass (FP) method (TFP) was compared with that measured by phantom method using an acrylic phantom in 45 patients with cardiac disease. Heart per mediastinum ratio (H/M) was compared to myocardial uptake calculated with TFP. The total injected dose measured using the phantom in which the syringe was set in depth of 3.5 cm (Tpham) was correlated with TFP (r = 0.73, p = 0.0001). When Tpham was corrected by body weight (c-Tpham), c-Tpham showed better correlation with TFP. MU calculated by TFP (MU-FP) was well correlated with MU by c-Tpham (MU-pham) (r = 0.94, p = 0.001). These results indicate that phantom method is sufficient to substitute for FP method. Though H/M was correlated with MU-FP (p < 0.001), the interpatient variation was relatively large. Then the analysis by H/M is insufficient to substitute for the myocardial uptake. It is though to be enough to use the phantom method on daily routine work, since this method is accurate and easy to quantitate the myocardial uptake of MIBG taking a short time.
3-Iodobenzylguanidine↗
[Artifactual defect of inferior myocardium on 123I-metaiodobenzylguanidine myocardial SPECT: characteristic findings and preventive method on phantom study].
Discordance between planar and SPECT image of inferior myocardial defect on 123I-metaiodobenzylguanidine myocardial scintigraphy (MIBG) was occasionally observed in the clinical studies. The purpose of this study is to clarify the existence of artifactual myocardial defect on MIBG SPECT image. Phantom study was performed in various kinds of situation using triple gamma camera. Artifactual defect adjacent to the liver was significantly observed in the case of increased liver/heart uptake ratio more than 2:1. Artifactual defect was markedly observed in the 180 degrees than that in the 360 degrees SPECT acquisition. To avoid artifactual defect during SPECT acquisition, liver phantom was moved down to the foot direction (vanishing liver position). In this phantom position, artifactual defect adjacent to the liver was not observed on reconstructed myocardial SPECT image. The vanishing liver position was also applicable to human study. Human body was inclined to right side at an angle of 15 degrees, artifactual defect could be avoided. Further study is needed to analyze the cause of this artifactual defect and to conform the preventive method with vanishing liver position in the routine myocardial SPECT examination.
3-Iodobenzylguanidine↗
Crystal structures of ribonuclease HI active site mutants from Escherichia coli.
In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.
Binding Sites↗
Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.
The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core.
The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.
Amino Acids↗
Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution.
The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution. The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity. Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances. Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes. The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands). However, some notable differences, which account for the enhanced thermostability of T. thermophilus RNase H, are observed in loop structures and side-chain conformations. The substitution of Gly for the left-handed helical residue (Lys95) in the E. coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom. Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.
Amino Acid Sequence↗
Role of histidine 124 in the catalytic function of ribonuclease HI from Escherichia coli.
The role of the conserved histidine residue (His124) in the catalytic function of Escherichia coli ribonuclease HI was probed by the use of the pH titration experiments with 1H NMR and site-directed mutagenesis. His124 is located close to the catalytic triad formed by the three carboxylates. The C2H proton resonance of His124, as well as those of four other His residues, in the enzyme were assigned by comparing the 1H NMR spectrum of the wild-type enzyme to the spectra of the five mutant enzymes, each of which lacked a different His residue. From the analysis of the pH titration shifts of these resonances, the pK alpha values of all His residues were determined. The pK alpha value of His124 was 7.1, which is slightly higher than, but close to, the normal value of the imidazole group. This result suggests that His124 is located in an acidic environment but does not interact directly with a carboxyl group in the solution structure. Three mutant enzymes, in which His124 was replaced by Lys, Gln, or Glu were constructed. The kinetic parameters of these mutant enzymes were determined by using the M13 DNA/RNA hybrid as a substrate. Substitution of Lys, Gln, or Glu for His124 dramatically lowered the Vmax value by 30-100-fold, without significantly affecting the Km value, as compared to those of the wild-type enzyme. In addition, any mutation impaired the in vivo function of the enzyme. These results indicate that His124 is involved in the catalytic function of the enzyme. We propose that His124 changes its conformation in the catalytic reaction and enhances the catalytic efficiency by removing a proton from a catalytically essential carboxylate.
Amino Acid Sequence↗
Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8.
Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with unit cell parameters a = b = 44.7 A, c = 314.7 A. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 A resolution.
Crystallization↗
Inhibition of epidermal growth factor-stimulated hepatocytes proliferation by autologous sinusoidal mononuclear cells in rat liver.
Mononuclear cells isolated from sinusoid of rat liver by collagenase perfusion method showed a strong inhibitory effect on epidermal growth factor-stimulated proliferation of cocultured autologous hepatocytes, while mononuclear cells from peripheral blood and spleen did not. This inhibitory effect depended on the effector/target ratio. In a single-color flow cytometric analysis, about 25% of sinusoidal mononuclear cells consisted of asialo GM1+ natural killer cells, and there were relatively small numbers of CD3+ T cells and a few leukocyte common antigen-positive B cells compared to peripheral blood mononuclear cells. The natural killer cell activity of sinusoidal mononuclear cells was much stronger than that of peripheral blood mononuclear cells and splenic mononuclear cells. This activity was completely suppressed by the treatment with anti-asialo GM1 antibody plus complement. However, their inhibitory activity on proliferation of autologous hepatocytes was not affected by this treatment. These results suggest that the sinusoidal mononuclear cells, probably CD3+ T cells, play a role in regulation of autologous hepatocyte proliferation.
Animals↗
pH-dependent thermostabilization of Escherichia coli ribonuclease HI by histidine to alanine substitutions.
Thermal stabilities of mutant ribonuclease HI proteins from Escherichia coli, in which each of five histidine residues was replaced with alanine, were examined at various pHs. Increases in the Tm values were observed at pH 3.0 for four of the mutant proteins, in which each of the four histidine residues exposed to the solvent was mutated, as compared to the Tm of the wild-type protein. The thermostabilization of three of the mutant proteins was dependent on pH, and only observed at low pH. The thermostabilizing effects of the His-->Ala substitutions were cumulative. The temperature of the midpoint of the transition in the thermal unfolding curves, Tm, of the most stable mutant enzyme, in which His 62, His 83, His 124, and His 127 were replaced by Ala, was 5.5 degrees C higher than that of the wild-type enzyme at pH 3.0. The stability of the wild-type protein decreased as the pH was lowered below pH 4, a condition favoring the protonation of carboxyl groups, probably due to unfavorable electrostatic interactions introduced by the increase in positive charges on the protein. Since imidazole groups are positively charged at pH 3.0, it seems likely that thermal stabilization at pH 3.0 by a His-->Ala substitution would be the result of a reduction in such unfavorable electrostatic interactions. These results suggest that amino acid substitutions that cause a decrease in the number of positive charges on the surface of a protein can be used as a general strategy to enhance protein stability at pH values below pH 4.
Alanine↗
Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability.
Systematic replacement of the amino acid residues in Escherichia coli ribonuclease HI with those in the thermophilic counterpart has revealed that two mutations, His62-->Pro (H62P) and Lys95-->Gly (K95G), increased the thermostability of the protein. These single-site mutant proteins, together with the mutant proteins His62-->Ala (H62A), Lys95-->Asn (K95N) and Lys95-->Ala (K95A), were crystallized and their structures were determined at 1.8 A resolution. The crystal structures of these mutant proteins reveal that only the local structure around each mutation site is essential for the increase in thermostability. For each mutant protein, the stabilization mechanism is considered to be as follows: (i) H62P is stabilized because of a decrease in the entropy of the unfolded state, without a change in the native backbone structure; (ii) K95G is stabilized since the strain caused by the left-handed backbone structure in the typical 3:5 type loop is eliminated; and (iii) K95N is slightly stabilized by a hydrogen bond formed between the side-chain N delta-atom of the mutated aspargine residue and the main-chain carbonyl oxygen within the same residue.
Enzyme Stability↗
[Cardiorespiratory responses during flume swimming and treadmill running in swimmers].
The present study was designed to compare cardiorespiratory responses of swimmers to swimming and running. Six male trained college swimmers performed maximal work test (progressive method) in flume swimming and treadmill running. VEmax during swimming (128.3 +/- 20.6 l/min) was about 4% lower than during running (133.2 +/- 9.9 l/min). VO2max during swimming (3628 +/- 228 ml/min) was significantly higher than during running (3408 +/- 222 ml/min). HRmax during swimming (191 +/- 8 beats/min) was significantly lower than during running (198 +/- 6 beats/min). VO2 at ventilatory threshold (VT) during swimming (2177 +/- 183 ml/min) was significantly higher than during running (1699 +/- 214 ml/min). %VO2max at VT during swimming (60.1 +/- 4.2%) was significantly about 10% higher than during running (49.8 +/- 4.4%). These results suggest that subjects of this study have specific cardiorespiratory adaptation to swimming from a long period of swim training.
Adult↗
[A case of pulmonary metastasis from colon cancer successfully treated by 5-FU combined with leucovorin and interferon alpha-2a].
A 58-year-old woman with colon cancer, who had received oral 5-FU over 17 months after right hemicolectomy, was diagnosed as having a recurrence of the disease with multiple pulmonary metastasis. She was treated for 5 days with a combination of continuous infusion of 5-FU 600 mg/m2/day, bolus injection of leucovorin (LV) 20 mg/m2/day, and intramuscular injection of interferon (IFN)-alpha-2a (6.0 x 10(6) U/day, repeated every 3 weeks. The chest X-ray after three cycles showed a decrease in size of metastatic lesions by 51%, indicating a partial response. Correspondingly, the serum levels of CEA and CA 19-9 significantly decreased. There were modest but tolerable side effects such as fever, nausea, diarrhea, stomatitis, and alopecia. The patient has been given oral UFT and LV after discharge, and is still alive with continued improvement of pulmonary lesions even 9 months after initial chemotherapy. Although the detailed synergistic mechanism of 5-FU and IFN has yet to be determined, the addition of IFN, as a biochemical modulator distinct from LV, to the combination of 5-FU and LV, appears to further potentiate the therapeutic efficacy and may be useful for advanced colorectal cancer.
Adenocarcinoma↗