Biomedical subjects

S Takai

Publications and source records attributed to S Takai.

At least 73 records · Page 4Linked to original sources

PubMed2000

Microstructural properties of the distal growth plate of the rabbit radius and ulna: biomechanical, biochemical, and morphological studies.

The purposes of this study were to define the tensile properties of each zone of the rabbit growth plate and to correlate them with the microarchitecture and biochemical composition of the zones. The epiphysis-growth plate-metaphysis complex was obtained from the radius and ulna of 20 8-week-old rabbits. Four dye markers were placed on the growth plate. The complex was loaded to failure with a tensile testing machine, and the strain behavior was recorded simultaneously with a microscope, a charge-coupled device camera, and a video dimension-analyzer system. The collagenous fiber architecture of each zone was examined with a microscope, and the collagen content of each zone was also determined. The tangent modulus of the resting zone was 75% stiffer than that of the other two zones. The highest values for strain at failure and energy absorbed to failure were observed in the hypertrophic zone, and the total collagen content was highest in the proliferating zone. The collagen fibers were more randomly aligned in the resting zone than in the other two zones. The diversity observed in the microarchitecture of the rabbit growth plate correlates with the zone-dependent differences in its mechanical properties.

Animals↗

PubMed2000

Association between the expression of mast cell chymase and intraperitoneal adhesion formation in mice.

BACKGROUND: Adhesion formation is a major source of postoperative morbidity and mortality. Mast cells and their major protease, chymase, have been shown to participate in the healing process as well as in tissue remodeling. We aimed to identify the role of mast cells in intraperitoneal adhesion formation and to assess whether there is an association between the expression of mast cell chymase and adhesion formation. MATERIALS AND METHODS: Both mast cell-deficient W/W(V) mice and congenic +/+ mice received a standardized lesion produced by cecal scraping and the application of 95% ethanol. Adhesions were assessed blindly 1 week later using a standardized scale. In addition, histamine content, mast cell numbers, and chymase activity in cecum as well as at the healing sites were evaluated before and 7 days after surgical injury. RESULTS: A significant reduction in adhesion formation was seen in mast cell-deficient W/W(V) mice (P < 0.05). In the normal cecum, histamine content did not significantly differ between W/W(V) and +/+ mice. Chymase activity in cecum was detected in control +/+ mice, but not in W/W(V) mice. Mast cell numbers and chymase activity levels at the healing sites of +/+ mice were significantly increased 7 days after surgery. CONCLUSIONS: Our results indicate that mast cells contribute to intraperitoneal adhesion formation in mice, and suggest that chymase originating from mast cells is important in the development of adhesions.

Animals↗

PubMed2000

Impact load transmission of the knee joint-influence of leg alignment and the role of meniscus and articular cartilage.

OBJECTIVE: The objective of this study was to evaluate load transmission in the cancellous bone of the tibia under static and impact load.BACKGROUND. Abnormal transmission of loads may result in osteoarthritis and fractures. However, the role of the cancellous bone in these processes is not well understood.METHODS. The compressive stresses in the subchondral bone, epiphysis and diaphysis of the tibia of porcine knees were measured under static and impact load using mini-pressure transducers. The tests were performed using a drop-tower type testing machine in neutral, varus and valgus alignments. Tests were repeated after meniscectomy and again after removing the articular cartilage.RESULTS. In the intact knee in all alignments, the highest stress on the medial side was found in the epiphysis, and in the subchondral bone on the lateral side. After meniscectomy, a significant increase was observed in the stress in the subchondral bone on both sides. After the articular cartilage was also removed, the stress in the subchondral bone increased again, but slightly.CONCLUSION. The importance of the meniscus in load transmission is supported by this study. The contribution of the articular cartilage to load transmission is less than that of the meniscus.Relevance. The patterns of static and dynamic load transmission observed in this study correlate with the sites of insufficiency fractures of the medial tibial plateau and traumatic lateral tibial plateau fractures observed clinically. Examination of the load transmission is key to a more complete understanding of the causes of osteoarthritis and tibial plateau fractures.

Analysis of Variance↗

PubMed2000

A new method for simultaneous measurements of mast cell proteases in human vascular tissue.

1. Human mast cells contain carboxypeptidase A, chymase and tryptase. In the present study, in order to analyse the mast cell proteases simultaneously, we investigated a method for the measurement of carboxypeptidase A, chymase and tryptase in human vascular tissues. 2. Human vascular tissues were homogenized in 10 mmol/L phosphate buffer containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 or 1.8 mol/L KCl and 0.1% non-idet P-40 and samples were then extracted. Because carboxypeptidase A and chymase convert angiotensin (Ang)I to Ang-(1-9) and AngII, respectively, the extracts were incubated with AngI in the presence of an angiotensin-converting enzyme (ACE) inhibitor. The extract prepared in buffer with over 0.8 mol/L KCl converted AngI to Ang-(1-9) and AngII. Formation of Ang-(1-9) and AngII plateaued in extracts with 1.0 and 1.2 mol/L KCl, respectively. 3. The formation of Ang-(1-9) and AngII in the extract with 1.2 mol/L KCl was inhibited by inhibitors of carboxypeptidase A and chymase, respectively, suggesting that Ang-(1-9) and AngII were generated from AngI by carboxypeptidase A and chymase, respectively. 4. Using a specific tryptase substrate, tryptase activity was detected in extract in buffer with over 0.8 mol/L KCl and reached a plateau at concentrations of KCl over 1.0 mol/L. 5. These findings show that the maximum activity of carboxypeptidase A, chymase and tryptase was detected in extracts of human homogenized vascular tissues in buffer at 1.2 mol/L KCl. The present study demonstrates a new method for the simultaneous measurement of proteases derived from mast cells in humans.

Angiotensin I↗

PubMed2000

Regulation of chymase production in human mast cell progenitors.

BACKGROUND: Although mature tryptase-positive mast cells (MCs) and tryptase and chymase double-positive MCs are recognized using in situ staining and are preferentially distributed in different tissues, recent findings suggest that tryptase-positive MCs can give rise to tryptase and chymase double-positive MCs. OBJECTIVE: We investigated the regulation of chymase production in developing MCs. METHODS: Human cord blood or peripheral blood cells were cultured in the presence of stem cell factor and IL-6 with or without IL-4 in methylcellulose or liquid medium. Intracellular chymase and tryptase were determined with immunocytochemistry, flow cytometry, and ELISA. Chymase messenger RNA expression was examined with 3 different methods, such as Northern blotting. RESULTS: Flow cytometric analysis always showed a unimodal histogram of chymase-positive, as well as tryptase-positive, cells in the presence of various cytokines, even when chymase was not detected in some MCs with immunocytochemistry. The chymase protein expression increased by culture duration and was enhanced by cytokines, such as a high concentration of stem cell factor or IL-4. Chymase messenger RNA was expressed higher in immature MCs than mature chymase protein-rich MCs. We generated macroscopic MC colonies in methylcellulose by culturing CD34(+) cells for 10 weeks and measured cellular chymase, tryptase, and histamine. The chymase/histamine ratio widely varied (0.07-1.01) depending on MC colony, even under the same culture conditions, including IL-4, whereas the tryptase/histamine ratio was relatively constant (1.02-1.89). CONCLUSION: All human MCs in culture are capable of producing chymase, and the production is clonally regulated at their progenitors by cytokine-independent mechanisms, as well as being totally controlled by cytokine-dependent mechanisms accompanied by maturation.

Cells, Cultured↗

PubMed2000

Bilateral epiphyseal fractures of the proximal tibia within a six-month interval: a case report.

An epiphyseal fracture commonly results from avulsion of the epiphysis by traction through the attached ligaments. A fracture of the proximal tibial epiphysis is, therefore, a rare injury because of the absence of collateral ligament attachments. Most proximal tibial epiphyseal injuries occur as avulsion fractures of the tibial tubercle; Salter-Harris Types I or II injuries involving pressure epiphyses are rare. In the current case of a thirteen-year-old boy, the proximal tibial epiphyseal injuries in both knees occurred during quadriceps contraction in the absence of violent trauma, and there was a six-month interval between the two injuries.

Adolescent↗

PubMed2000

DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103.

The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.

Amino Acid Sequence↗

PubMed2000

Characterization of recombinant human chymase expressed in Escherichia coli.

We compared recombinant human chymase expressed in Escherichia coli with human chymase purified from vascular tissues. The recombinant chymase, the structure of which was NH2-enterokinase cleavage site-chymase-COOH, was expressed in Escherichia coli and then was solubilized and renatured. The protein did not have a chymase activity, but gained this activity after the cleavage of the N-terminal site by enterokinase. The enzyme was purified by heparin affinity and gel filtration columns. The N-terminal sequence of the protein was identical to the sequence for human chymase. The molecular weights of the recombinant chymase and chymase purified from human vascular tissues were 26 and 30 kDa, respectively, and the 4 kDa difference was thought to be due to the presence or absence of glycan. The optimum pH of the recombinant enzyme activity was between 7.5 and 9.0. The activity of the recombinant enzyme was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by ethylenediaminetetraacetic acid and aprotinin. This enzyme cleaved specifically the Phe8-His9 bond of angiotensin (Ang) I to form Ang II and that of big endothelin (ET)-1 to form ET-1-(1-31). These findings demonstrated that the enzymatic characteristics of the recombinant enzyme were identical to that of native human chymase.

Amino Acid Sequence↗

PubMed2000

Characteristics of monkey tryptase purified from cheek pouch vascular tissues.

Tryptase purified from rat and dog tissues has been reported, although the characteristics of these enzymes are different from human tryptase. For pathophysiological studies of human tryptase, studies on species that have a similar tryptase to humans is needed. In this study, we purified monkey tryptase from cheek pouch vascular tissues using heparin affinity and gel filtration columns. The monkey tryptase, which had a molecular weight of 130 kDa by gel filtration, consisted of a tetramer of 33 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal sequence showed high homology with tryptases from other species. The optimum pH and temperature were 7.5-9.0 and 25-40 degrees C, respectively. The enzyme was labile in high-KCl buffer, and the optimum KCl concentration was 0.1 M. The enzyme activity was completely inhibited by diisopropyl phosphorofluoridate and leupeptin but not by soybean trypsin inhibitor and alpha-antitrypsin. The enzyme hydrolyzed vasoactive intestinal peptide but did not affect angiotensin I, somatostatin and bradykinin. In the present study, we first isolated monkey tryptase from cheek pouch vascular tissues and showed that the characteristics of monkey tryptase are very similar to those of human tryptase.

Animals↗

PubMed2000

The functional ratio of chymase and angiotensin converting enzyme in angiotensin I-induced vascular contraction in monkeys, dogs and rats.

Recently, a chymase-dependent angiotensin (Ang) II-forming pathway was found in human cardiovascular tissues, and the significance of this pathway in the pathogenesis of some cardiovascular diseases was suggested. The present study examined the ratio of angiotensin converting enzyme (ACE) to chymase-dependent Ang II formation in various isolated vessels from monkeys, dogs and rats. In all of the examined vessels, the addition of KCl at a concentration of 50 mM could induce a maximal contraction. Except for monkey coronary artery and rat renal and femoral artery, the addition of Ang I could induce transitory contractions, whereas the force of contractions in these vessels was quite different. The sensitivity to Ang II in these vessels was similar to that for Ang I. In monkey gastroepiploic and mesenteric arteries, about 70% of the Ang I-induced contraction was suppressed by chymase inhibition, while it was suppressed about 50% in monkey renal, femoral and carotid arteries. In dog renal arteries, about 65% of the Ang I-induced contraction was suppressed by chymase inhibition, while it was suppressed by about 30% in other dog arteries. In contrast, in all rat arteries, Ang I-induced contractions were completely suppressed by treatment with ACE inhibitor alone. We concluded that regional differences in the response to Ang I exist in vascular tissues, and the ratio of ACE- to chymase-dependent Ang II formation is different in the various vessels.

Angiotensin I↗

PubMed2000

Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals.

The reliability of preparing bacteriological cultures from nasotracheal aspirates of foals routinely in order to diagnose R. equi pneumonia in foals was studied by isolating Rhodococcus equi from specimens obtained from 96 foals by nasotracheal aspiration with a silicon catheter. Results were compared with specimens obtained from 21 foals by transtracheal aspiration (percutaneous tracheal puncture). These 117 foals showed clinical signs of respiratory tract infection at sampling. R. equi was isolated from 14 of 21 (66.7%) specimens by transtracheal aspiration and from 59 of 96 (61.4%) specimens by nasotracheal aspiration, 649 of 655 isolates (99.1%) from the 73 positive specimens were virulent R. equi, and the culture-positive foals were diagnosed as having R. equi pneumonia. To assess the contamination of aspirates by organisms from the nasopharynx, the results of R. equi isolation from nasal swabs obtained from 56 of the 96 foals were compared to those obtained by nasotracheal aspiration from the same foals. R. equi was isolated from 2 of the 56 nasal swabs: one from a tracheal aspirate was positive, and the other was not. These results suggest that the nasotracheal aspiration technique, which is noninvasive and not associated with complications, could be used as an alternative to the transtracheal aspiration method, especially for the diagnosis of R. equi pneumonia in foals.

Actinomycetales Infections↗

PubMed2000

Role of chymase on vascular proliferation.

In the normal state, vascular ACE regulates local angiotensin II formation and plays a crucial role in the regulation of blood pressure, whereas chymase is stored in secretory granules in mast cells and has no enzymatic effects such as angiotensin II-forming activity. Chymase has a maximal activity immediately upon release into the extracellular matrix in vascular tissues after mast cells have been activated by a strong stimulus such as experienced by catheter-injured and grafted vessels. Therefore, chymase plays an important role in forming local angiotensin II when vascular tissues are injured, and inhibition of chymase may be useful for preventing vascular proliferation in grafted vessels and after PTCA (Figure 6).

Angiotensin II↗

PubMed1999

Chymase-dependent angiotensin II formation in human vascular tissue.

BACKGROUND: Some reports have suggested that, in vitro, human heart chymase in homogenates contributes little to angiotensin (Ang) II formation in the presence of natural protease inhibitors such as alpha-antitrypsin. We studied whether chymase bound to heparin, resembling an in vivo form, could contribute to Ang II formation in the presence of natural protease inhibitors. METHODS AND RESULTS: The Ang II formation was increased time-dependently after incubation in an extract (1 mg of protein/mL) of human vascular tissues containing Ang I. The concentration of Ang II in the extract after incubation for 30 minutes was 1.67+/-0.06 nmol/mL, and we regarded this quantity of Ang II as 100%. The Ang II formation was inhibited 10%, 95%, and 96% by 1 micromol/L lisinopril, 100 micromol/L chymostatin, and 0.1 g/L alpha-antitrypsin, respectively. The extract was applied to a heparin affinity column. After the column was washed with PBS, the eluted PBS contained a weak Ang II-forming activity, which was completely inhibited by lisinopril. The eluted PBS, to which >0.8 mol/L NaCl had been added, showed a strong Ang II-forming activity which was inhibited by chymostatin and alpha-antitrypsin. After the application of the extract, the column was washed with PBS and then an Ang I solution in PBS was applied to the column. The Ang II formation in the PBS eluted from the incubated column was increased time-dependently. The concentration of Ang II in the PBS (1 mL) eluted from the column after incubation for 30 minutes was 2.56+/-0.28 nmol/mL, and we regarded this quantity of Ang II as 100%. To study the effects of inhibitors, the extract (1 mg of protein/mL) was applied to a heparin affinity column (1 mL) which was preequilibrated with PBS (3 mL); 100 micromol/L chymostatin or 0.1 g/L alpha-antitrypsin in PBS (1 mL) was then applied to the column. After the column was washed with PBS (3 mL), Ang I solution (1 mg/mL) in PBS was applied to the column, and the column was incubated for 30 minutes. The Ang II formation in the PBS eluted from the column was suppressed up to 5% by application of chymostatin, although this was not affected by application of alpha-antitrypsin. CONCLUSIONS: These findings suggest that human chymase bound to heparin plays a functional role in Ang II formation in the presence of natural protease inhibitors such as alpha-antitrypsin.

Adult↗

PubMed1999

A novel type of non-coding RNA expressed in the rat brain.

We have characterized a novel type of non-coding RNA which consists of tandem repeats of similar sequences, approximately 0.9 kb in size. This RNA, termed Bsr (brain specific repetitive) RNA, is encoded at a single locus (6 q31-->q32) in the rat genome, where 100 to 150 copies of the 0.9 kb sequences are repeated in tandem. Bsr RNA is preferentially expressed in the rat central nervous system (CNS), especially in phylogenetically old structures, such as the pareo- and archicortex, amygdala, thalamus and hypothalamus. In the developing brains, Bsr RNA is expressed in the subsets of differentiating cells but not in proliferating cells. Despite the finding that Bsr RNA appears to be conserved only among the Rattus species, the specific expression pattern of Bsr RNA suggests that it might have some role in the rat CNS.

Animals↗

PubMed1999

Lack of effect of carbohydrate depletion on some properties of human mast cell chymase.

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This enzymatic deglycosylation product was enough to explore possibilities that N-glycan might modify some properties of human chymase. Substrate specificity, optimum pH and the elution profile from the heparin affinity gel were not affected by the deglycosylation. Only a slight but significant difference was observed in the Km value for conversion of angiotensin I to angiotensin II. Other kinetic constants such as kcat were not influenced. The kinetics of conversion of big endothelin-1 to endothelin-1(1-31) were not significantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline pH and thermal stress. Even at physiological temperature and pH, the activity of glycosylated human chymase was more stable. From these results, it appears that the N-glycan of human chymase contributes to the stability of this enzyme but not to its functional properties.

Carbohydrates↗

PubMed1999

Tranilast suppresses vascular chymase expression and neointima formation in balloon-injured dog carotid artery.

BACKGROUND: Activation of vascular chymase plays a major role in myointimal hypertrophy after vascular injury by augmenting the production of angiotensin (ANG) II. Because chymase is synthesized mainly in mast cells, we assumed that the chymase-dependent ANG II formation could be downregulated by tranilast, a mast cell-stabilizing antiallergic agent. We have assessed inhibitory effects of tranilast on neointima formation after balloon injury in the carotid artery of dogs, which share a similar ANG II-forming chymase with humans, and further explored the pathophysiological significance of vascular chymase. METHODS AND RESULTS: Either tranilast (50 mg/kg BID) or vehicle was orally administered to beagles for 2 weeks before and 4 weeks after balloon injury. Four weeks after the injury, remarkable neointima was formed in the carotid arteries of vehicle-treated dogs. Chymase mRNA levels and chymaselike activity of vehicle-treated injured arteries were increased 10.2- and 4.8-fold, respectively, those of uninjured arteries. Angiotensin-converting enzyme (ACE) activity was slightly increased in the injured arteries, whereas ACE mRNA levels were not. Tranilast treatment completely prevented the increase in chymaselike activity, reduced the chymase mRNA levels by 43%, and decreased the carotid intima/media ratio by 63%. In vehicle-treated injured arteries, mast cell count in the adventitia showed a great increase, which was completely prevented by the tranilast treatment. Vascular ACE activity and mRNA levels were unaffected by tranilast. CONCLUSIONS: Tranilast suppressed chymase gene expression, which was specifically activated in the injured arteries, and prevented neointima formation. Suppression of the chymase-dependent ANG II-forming pathway may contribute to the beneficial effects of tranilast.

Animals↗

PubMed1999

Ovarian carcinoma with fistula formation to the sigmoid colon and ileum: report of a case.

We describe herein an extremely rare case of clear cell type ovarian carcinoma resulting in fistula formation into the colon and intestine. The patient was a 61-year-old woman in whom a large tumor with extravasation from the sigmoid colon was found by barium enema examination. The tumor was preoperatively diagnosed as left ovarian cancer by angiography which showed the tumor feeder arising from the left ovarian and uterine arteries.

Adenocarcinoma, Clear Cell↗

PubMed1999

Unusual proximal tibiofibular synostosis.

Proximal tibiofibular synostosis without multiple hereditary exostosis is extremely rare and only 7 cases have been reported in the literature. All of the previously reported cases accompanied deformities such as distal positioning of the proximal tibiofibular joint, leg length discrepancy, bowing of the fibula, and valgus deformity of the knee. The present case of a 24-year-old man had neither a history of trauma nor deformity around the knee. Therefore, it was suggested that this type of synostosis occurred after epiphyseal plate closure.

Adult↗