Biomedical subjects
Publications and source records attributed to S Terada.
A new method was developed for measuring the ATP regeneration rate by creatine kinase from creatine phosphate and ADP. The ATP regeneration rate was calculated to be 0.20 mM x s(-1) at room temperature from the pH change of the regeneration mixture.
Two platelet aggregation inhibitors, ussuristatin 1 (US-1) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Mrs of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer. Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50 = 17-33 nM. US-2 also inhibited the platelet aggregation, but the IC50s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.
A non-hemorrhagic proteinase, brevilysin L6 (L6), has been purified to homogeneity from Agkistrodon halys brevicaudus venom by gel filtration and DEAE-Toyopearl 650M chromatography. It is an acidic protein with an isoelectric point of 4.8, and its molecular mass was estimated to be 21.5 kDa by SDS-PAGE. The optimum pH of L6 was about 9. EDTA and o-phenanthroline inhibited the proteolytic activity, suggesting that L6 is a metalloprotease. Cysteine also inhibited the activity of L6, but glutathione did not. The protein was stable in the pH range of 5-8.5 and below 40 degreesC. Calcium ions had no effect on the proteolytic activity of L6 or on its thermal stability. The enzyme preferentially cleaved X-Leu, X-Phe, X-Val, and X-His bonds. L6 showed weak alpha-fibrinogenase activity. The complete amino acid sequence of L6 was also determined by manual Edman degradation. L6 is a non-glycosylated single-chain polypeptide consisting of 203 residues with an N-terminal pyroglutamic acid and a calculated molecular weight of 22,713 Da. Its entire sequence is highly homologous to those of other metalloproteases from various snake venoms. A zinc-binding motif, HEXXHXXGXXH, is located at residues 143-153 in the sequence of L6.
Single items from a typical clinical examination have proved disappointing in their predictive value for temporomandibular joint (TMJ) disc displacement. Only one criterion (the 12 o'clock) is used to diagnose normal disc position. According to this criterion, the posterior band of the disc should be located at the top of the condyle, at the 12 o'clock position. The purpose of this study was to determine which signs and symptoms provide a valid prediction of the condition of the joint based on 4 magnetic resonance imaging (MRI) criteria used to define normal disc position. Sagittal MRI and clinical findings of 137 temporomandibular disorder patients and 23 normal asymptomatic volunteers were used. Three calibrated and blinded observers interpreted the images. Disc position with the mouth closed was evaluated based on 4 MRI criteria: 12, 11, 10 o'clock, and the intermediate zone. Disc position with the mouth open was determined based on one criterion. It was considered normal if the intermediate zone of the disc was located between the condyle and the articular eminence. Joints were classified as normal or as having disc displacement with or without reduction. The sensitivity and specificity of multiple clinical parameters for predicting the condition of the joint established by each of these 4 gold-standard MRI criteria were then determined. Regarding disc displacement with reduction, significant differences were observed in the sensitivity and specificity of all of the clinical parameters used to predict the imaging diagnosis established by each of the criteria. Concerning disc displacement without reduction, no significant differences were observed. The intermediate zone criterion was the criterion that most accurately reflected the condition of the joint. The clinical predictability of the disorder diagnosed according to this criterion suggests that clinical findings alone are too often nonspecific as predictors of the imaging stage of disc displacement. However, we found that combining the most sensitive clinical items to predict the disorder and using an overall criterion for positivity to interpret the results led to an impressive increase in the specificity of the combination, enabling false-positive diagnoses to be excluded.
The effects of high pressure (0.1-80 MPa) on cell adhesion were examined using Chinese hamster ovary cells. The cells detached by EDTA or trypsin were cultured for 4 h at atmospheric pressure. In each case, the rate of the adhesive cells was about 85%. Upon the addition of cycloheximide, such cell adhesion was almost completely inhibited in trypsin-treated cells but less affected in EDTA-treated cells. When the EDTA-detached cells were subjected to high pressures and cultured at atmospheric pressure, the cell adhesion was unaffected up to 40 MPa, but was suppressed significantly at higher pressures although the integrins were expressed on the plasma membranes. The suppressive effects induced by high pressure (80 MPa) were marked in trypsin-treated cells, in which the integrins were digested. Furthermore, actin stress fibers in spread cells disappeared due to cell rounding upon exposure to 80 MPa, and such rounded cells were partially detached from the substratum. These results suggest that the retardation of adhesion by high pressure in EDTA-detached cells may be due to the perturbed interactions between integrin and its associated proteins, whereas that of trypsin-detached cells is due to the delayed expression of integrins toward the membrane surface, in addition to the cause seen in the case of EDTA.
The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.
F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 10(6) cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.
Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.
A pregnant woman with transient gastric obstruction was fed by enteral nutrition alone for 11 wk and 6 d. Her body mass index of 23.2 kg/m2 before pregnancy declined to 21.8 kg/m2 after delivery. Although she did not gain weight during enteral nutrition, fetal growth, estimated by ultrasonography, was normal, and she delivered a 3030-g female infant at 38 wk gestation. The blood laboratory data during pregnancy and the breast milk after pregnancy were also normal. The results suggest that factors other than maternal weight gain alone should be considered in the evaluation of nutritional status for the pregnant woman.
OBJECTIVE: This study was undertaken to evaluate different criteria to establish normal disk position on magnetic resonance images. STUDY DESIGN: Magnetic resonance image findings of 137 consecutive patients with temporomandibular disorders and 23 asymptomatic volunteers were used in this study. Three calibrated observers interpreted the images individually. Four closed-mouth and 1 open-mouth criteria were tested for their ability to define normal and abnormal temporomandibular joint disk positions on magnetic resonance images. RESULTS: For the 46 joints in the asymptomatic volunteers, the criterion that yielded the highest percentage of normal disk position diagnoses was the disk's intermediate zone (93.5%). Clock face criteria produced the following declining percentages of normal disk position diagnoses: 10 o'clock, 82.6%; 11 o'clock, 63.0%; and 12 o'clock, 39.1%. Similar results were obtained for the patients with temporomandibular disorders. In both groups, as the number of normal disk position diagnoses declined, the percentage of joints with a diagnosis of disk displacement with reduction increased. Conversely, the percentage of joints with a diagnosis of disk displacement without reduction (in the group of patients with temporomandibular disorders) did not appear to be substantially affected by the 4 closed-mouth disk position criteria. CONCLUSIONS: These results suggest that the intermediate zone criterion for disk displacement is the more stringent criterion and the one that would yield the lowest number of false positives when the disk position is being judged in the closed-mouth sagittal view.
The relationship between signs and symptoms of temporomandibular disorders (TMD) and bilateral occlusal contact patterns was investigated in 143 TMD patients (mean age: 34.0 +/- 15.9 years; 38 male and 105 female). In addition to an interview regarding chief complaints and accompanying symptoms, various muscles and the temporomandibular joints were palpated bilaterally and occlusal analyses were made. Only 5 out of 108 paired variables were found to be significantly associated by using the chi-squared test. Medial pterygoid muscle pain on palpation showed significant associations with the occlusal contact pattern (P < 0.005), especially working side contacts (interocclusal tooth contacts on the working side) (P < 0.005), during contralateral excursions; sternocleidomastoid muscle pain on palpation showed a significant association with balancing side contacts (interocclusal tooth contacts on the balancing side) during ipsilateral excursions P < 0.05); shoulder stiffness and pain in the eye showed significant associations with balancing side contacts during contralateral excursions (P < 0.05). The results show only a weak relationship between some TMD symptomatology and bilateral occlusal contact patterns during lateral excursions. The findings suggesting the specific laterality of a few TMD signs and symptoms associated with particular occlusal contacts may deserve closer case-control study.
The effect of high pressure on proliferation and cell cycle progression was examined using murine erythroleukemia (MEL) cells. The MEL cells were exposed to high pressures (0.1-130 MPa) and then cultured for 5 days at atmospheric pressure. The proliferation of MEL cells was unaffected up to 60 MPa, but was suppressed at 80-110 MPa. Above 120 MPa, the cells were fragmented. The cell cycle analysis of 80 MPa-treated MEL cells showed that the cells in S phase are most sensitive to high pressure and they arrest in G2 phase. Interestingly, G2-arrested cells reinitiated DNA synthesis, resulting in giant cells with high DNA contents. Furthermore, when such G2-arrested cells were exposed to caffeine, premature mitosis, characterized by chromosome pulverization, was observed. These results suggest that the suppression of proliferation in high-pressure-treated MEL cells is associated with G2 arrest following S phase delay. Thus, it seems valuable to apply high pressure to the investigation of the cell cycle.
In the membrane stability of human erythrocytes, the role of two cysteine residues (Cys-201 and Cys-317) in the cytoplasmic domain of band 3 is not clear. So we tried to resolve this problem by examining hemolytic properties under high pressure. From SH contents and spin labeling, it was found that Cys-201 and Cys-317 of band 3 were modified with N-ethylmaleimide (NEM). The hemolysis of intact erythrocytes at 200 MPa was suppressed by the binding of 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), anion transport inhibitor, to band 3. Similarly, the suppressive effect of DIDS was observed in the erythrocyte that Cys-201 and Cys-317 were modified with NEM. These results suggest that the cysteine residues in the cytoplasmic domain of band 3 are not essential for the DIDS-induced membrane stabilization.
Increased pulmonary 201TlCl (Tl) uptake during exercise has been used as a marker of multivessel critical stenosis. We studied whether pulmonary 99mTc-Tetrofosmin (TF) uptake measurement during exercise is useful as an additional indicator for the detection of coronary artery disease. Pulmonary to myocardial uptake ratio (P/M) measured by TF scintigraphy during exercise were compared with findings of coronary angiography in eighty one patients with ischemic heart disease and also P/M measured with Tl in twenty one cases. TF P/M level in the patients with triple vessel disease was higher than that in the patients with no coronary stenosis, single vessel disease and double vessel disease. However, there was no significant correlation between TF P/M and the severity of coronary artery stenosis. Inverse correlation was observed between TF P/M and left ventricular ejection fraction (LVEF) (r = 0.29, p < 0.01). TF P/M in the patients less than 50% of LVEF was significantly higher than that in the patients over 50% of LVEF (p = 0.05). TF P/M was well correlated with Tl P/M (r = 0.86). In conclusion, quantitative TF P/M during exercise was thought to be useful indicator for the evaluation of coronary artery disease.
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In minilaparotomy-assisted vaginal hysterectomy, the operation begins vaginally by opening the peritoneal folds and ligating the uterine vessels and uterosacral ligaments. Minilaparotomy is then performed for myomectomy, cutting the fallopian tubes and the utero-ovarian ligaments and detaching any adhesions on the anterior peritoneal angle. In 26 women who underwent this procedure, the feasibility rate was 100% and no intraoperative complications or postoperative morbidity was observed (except in one case of ovarian bleeding), indicating that vaginal hysterectomy assisted by minilaparotomy is a feasible approach for hysterectomy in the setting of large myomas, myomas with adhesions caused by endometriosis or previous pelvic surgery, and adenomyosis.
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