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Biomedical subjects

Y Yamauchi

Publications and source records attributed to Y Yamauchi.

At least 19 recordsLinked to original sources

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment.

A chondrogenic cell line, TC6, was established by using cells derived from articular cartilage of transgenic mice harboring a temperature-sensitive mutant simian virus (SV) 40 large T-antigen gene. TC6 cells express genes encoding proteins related to cartilage phenotypes such as type II collagen. To examine the in vivo behavior of the TC6 cells, these cells were implanted into cavity-shaped full-thickness defects made in the articular cartilage of the central part of the patellar grooves of mouse femora. One week after implantation, the morphology of the cells was still fibroblastic but these cells were just about to start to form a cartilage-like matrix. By 6 weeks after implantation, the cells had produced abundant cartilaginous matrix and their morphology became closer to that of authentic chondrocytes. This was in sharp contrast to the fibroblastic morphology of these cells in an in vitro environment even after long-term culture. These observations indicate that a cartilage-matrix environment provides a scaffold for the TC6 cells to form cartilage tissues. Our data show that the genetically engineered chondrocytic cell line, TC6, can form a cartilage-like matrix in vivo.

Animals

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.

Animals

Rapid analysis of methylated xanthines in teas by an improved high-performance liquid chromatographic method using a polyvinylpolypyrroridone pre-column.

We have developed a new high-performance liquid chromatography (HPLC) method for the analysis of methylated xanthines in tea by removing polyphenols using a polyvinylpolypyrroridone (PVPP) pre-column. The PVPP pre-column was connected with the upstream of analytical column to remove catechins in tea extract. Using this pre-column, caffeine and theobromine in tea, which belong to methylated xanthines, could be rapidly determined in less than 10 min with an isocratic solvent system. RSDs of standard solutions of caffeine and theobromine were about 0.3 and 0.3% for the retention time, and were about 1.6 and 2.5% for the peak area. The quantitation curves of caffeine and theobromine were linear from 5 to over 1000 ng.

Chromatography, High Pressure Liquid

Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain.

Postnatal development, such as synapse refinement, is necessary for the establishment of a mature and functional central nervous system (CNS). Using differential display analysis, we identified a novel gene, termed Bdm1, that is more abundantly expressed in the adult brain than in the embryonic brain. The full-length Bdm1 cDNA is 2718 base pairs long and contains an open reading frame of 1059 base pairs encoding a 38-kDa protein. Northern blot analysis revealed that expression of Bdm1 mRNA in the brain was weak on embryonic days and increased in the early postnatal period. Bdm1 mRNA was significantly expressed in the brain and heart, but there was no or little expression in other tissues. During the differentiation of mouse carcinoma cells P19 to neuron-like cells by retinoic acid, Bdm1 mRNA was up-regulated almost parallel to neurofilament mRNA. Expression of Bdm1 mRNA was observed appreciably in PC12 cells after neuronal differentiation but not in the nonneural cell lines examined. In situ hybridization demonstrated that Bdm1 was expressed widely in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata. Taken together, these data suggest that Bdm1 gene plays a role in the early postnatal development and function of neuronal cells.

Amino Acid Sequence

Design of serine protease inhibitors with conformation restricted by amino acid side-chain-side-chain CH/pie interaction.

A novel type of conformationally restricted peptides with the structure of H-D-Xaa-Phe-NH-CH2-C6H5 has been developed as inhibitors of serine proteinase chymotrypsin. The D-Xaa-alkyl and Phe-phenyl groups resulted in a formation of the hydrophobic core due to the side-chain-side-chain CH/pie interaction. Their spatial proximity was evidenced by 400 MHz 1H-nmr measurements, observing large upfield shifts of proton signals of D-Xaa-alkyl and nuclear Over-hauser effect (NOE) enhancements between the D-Xaa-alkyl and Phe-phenyl groups. This conformational restriction brought by CH/pie interaction produced an inhibitory structure, in which the C-terminal amide-benzyl group fits the chymotrypsin S1 site and the hydrophobic core binds to the S2 site. The inhibitory conformation was demonstrated crystallographically for the complex between the dipeptide H-D-Leu-Phe-NH-CH2-C6H4(p-F) and gamma-chymotrypsin. Detailed structure-activity studies have substantiated the structure of dipeptides in the active center of the enzyme.

Binding Sites

Phenolic antioxidants from the leaves of Corchorus olitorius L.

Six phenolic antioxidative compounds [5-caffeoylquinic acid (chlorogenic acid), 3,5-dicaffeoylquinic acid, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-(6-malonylglucoside), and quercetin 3-(6-malonylgalactoside) (tentative)] were identified from the leaves of Corchorus olitorius L. (moroheiya) by NMR and FAB-MS. The contents of these phenolic compounds, ascorbic acid, and alpha-tocopherol in C. olitorius leaves were determined, and their antioxidative activities were measured using the radical generator-initiated peroxidation of linoleic acid. The results obtained showed that 5-caffeoylquinic acid was a predominant phenolic antioxidant in C. olitorius leaves.

Antioxidants

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

Amino Acid Sequence

Type A behavior is associated with an increased risk of left ventricular hypertrophy in male patients with essential hypertension.

OBJECTIVE: To determine whether type A behavior, which is associated with a risk of coronary heart disease, affects left ventricular hypertrophy in patients with essential hypertension. DESIGN: Cross-sectional study of 88 untreated patients with mild to moderate essential hypertension (33 men, mean +/- SEM age 54 +/- 1 years). METHODS: We measured the type A behavior score using a standardized questionnaire, left ventricular mass index using M-mode echocardiography and 24 h mean ambulatory blood pressure (recorded every 30 min). Beat-to-beat blood pressure was also measured using a Finapres device in patients at rest and during mental stress (counting backward) to determine the blood pressure response to stress. RESULTS: The left ventricular mass index was correlated with the type A behavior score (r = 0.214, P < 0.05), age (r = 0.266, P < 0.05), 24 h mean systolic and diastolic blood pressures (r = 0.391, P < 0.001, and r = 0.382, P < 0.001, respectively), systolic blood pressure both at rest and during stress (r = 0.255, P < 0.05, and r = 0.215, P < 0.05, respectively), and the variability of both systolic and diastolic blood pressures at rest (r = 0.253, P < 0.05, and r = 0.321, P < 0.01, respectively). Stepwise multiple linear regression analysis demonstrated that age was associated with an increase in the left ventricular mass index for both sexes (P = 0.004 for males, P = 0.003 for females). The type A behavior score predicted a greater increase in left ventricular mass index in men (P = 0.018) but not in women. The 24 h mean systolic blood pressure was associated with a greater increase in left ventricular mass index in women (P < 0.001) but not in men. CONCLUSION: Type A behavior is an independent risk factor for left ventricular hypertrophy in male patients with essential hypertension.

Blood Pressure

Real-time 3-D model-based navigation system for endoscopic paranasal sinus surgery.

This paper presents a three-dimensional (3-D)-model-based navigation system for endoscopic sinus surgery treating paranasal sinusitis. Endoscopic surgery is becoming more common because of its low invasiveness. Its problem with disorientation, however, is one of the toughest barriers for the novice and may lead even an expert to commit serious surgical errors, e.g., causing cerebrospinal fluid (CSF) leakage or blinding the patient. To prevent such complications and optimize training, our system aids in navigation by showing a single perspective view of the patient and the endoscope models. This virtual endoscope has a viewing cone with a simulated light to indicate the viewing direction and visual field in real-time. The system's three clipping planes automatically follow the endoscope and help keep the surgeon aware of the endoscope's actual position. An experiment comparing the system to conventional navigation using a triplanar display showed that the perspective view was referenced very frequently, giving a positive impressions, while the triplanar display was almost completely ignored, apparently because it was too difficult to interpret immediately.

Adult

C-Reactive protein in dilated cardiomyopathy.

The prognosis for patients with idiopathic dilated cardiomyopathy (DCM) is poor, although clinical features are variable. Prediction of outcome has been difficult in individual patients based on laboratory data. In some patients with DCM, myocardial damage secondary to viral or immune-mediated myocardial inflammation may persist. To objectively assess inflammation, we measured plasma concentrations of C-reactive protein (CRP) in 188 patients with idiopathic DCM over 5-8 years. All had dyspnea and fatigue at rest; all patients had a left ventricular ejection fraction less than 40% by echocardiography or by contrast or radionuclide ventriculography. We divided these patients into two groups: patients dying within 5 years following admission (n = 49) and the remainder surviving for at least 5 years (n = 139). CRP concentrations in the patients dying early were significantly higher than in the long-term survivors (1. 05 +/- 1.37 vs. 0.49 +/- 1.04 mg/dl, p < 0.05). Sixty-two percent of the patients with CRP>1.0 died within 5 years. In addition to other laboratory tests including electrocardiography and echocardiography, routine CRP measurements proved to be valuable for identifying high-risk patients who require special treatment strategies.

Adult

Clinical implications of lymph node micrometastases in patients with colorectal cancers. A case control study.

There is no unanimity about the prognostic significance of lymph node micrometastases from colorectal cancer. A case-control study of patients with recurrent and nonrecurrent colorectal cancer who were closely matched for the Dukes stage, extent of lymph node dissection, tumor size, tumor location, number of resected lymph nodes, age and gender was performed. The presence of micrometastases in a total of 1,633 lymph nodes from 44 patients (22 with and 22 without recurrence) were examined by immunohistochemistry using antibodies for cytokeratin (KL-1) and p53 (RSP53). Immunostaining with KL-1 revealed micrometastases in 15/22 patients [68%; 82/820 lymph nodes (10%)] and 15/22 patients [68%; 45/813 lymph nodes (6%)] in the recurrent and nonrecurrent groups, respectively. Immunohistochemical analysis, using RSP53, of 18 paired patients with p53-positive primary tumor revealed micrometastases in 4/9 patients [44%; (7/265 lymph nodes (2.6%)] and 4/9 patients [44%; 6/257 lymph nodes (2.3%)] with and without recurrence, respectively. Neither the micrometastatic frequencies of the patients nor the resected lymph nodes of the recurrent and nonrecurrent groups differed significantly. Micrometastases in regional lymph nodes are an interesting phenomenon, but do not influence patients' prognoses if the involved lymph nodes are removed.

Adult

A new model rat with acute bronchiolitis and its application to research on the toxicology of inhaled particulate matter.

The aim of the present study was to establish a useful animal model that simulates humans sensitive to inhaled particulate matter (PM). We have developed a new rat model of acute bronchiolitis (Br) by exposing animals to NiCl2 (Ni) aerosols for five days. Three days following the Ni exposure, the animals developed signs of tachypnea, mucous hypersecretion, and bronchiolar inflammation which seemed to progress quickly during the fourth to fifth day. They recovered from lesions after four weeks in clean air. To assess the sensitivity of the Br rats to inhaled particles, two kinds of PM of respirable size were tested with doses similar to or a little higher to the recommended threshold limit values (TLVs) for the working environment in Japan. Titanium dioxide (TiO2 = Ti) was chosen as an inert and insoluble particles and vanadium pentoxide (V2O5 = V), as a representative soluble and toxic airborne material. The Br rats exposed to either Ti or V were compared the pathological changes in the lungs and the clearance of particles to those in normal control or Br rats kept in clean air. The following significant differences were observed in Br rats: 1. delayed recovery from pre-existing lesions or exacerbated inflammation, 2. reductions in deposition and clearance rate of inhaled particles with the progress of lesions. The present results suggest that Br rats are more susceptible to inhaled particles than control rats. Therefore, concentrations of particulate matter lower than the TLVs for Japan, which have no harmful effects on normal lungs, may not always be safe in the case of pre-existing lung inflammation.

Acute Disease

[Acute otitis media associated with bone conduction hearing loss].

Eleven patients (12 ears) with acute otitis media associated with raised bone conduction were studied. The patients were from 19 to 70 years old and came to our hospital between November 1996 through May 1997. Pure tone audiometry revealed mixed hearing loss, but there was no bullous myringitis in all cases. They were treated by oral or intravenous antibiotics, steroids, and myringotomy. Bacteriological examination was done in seven cases, and revealed penicillin resistant Streptococcus pnumoniae in three cases. Complete recovery of hearing loss was obtained in 10 of 12 ears, but sensorineural hearing loss remained in 2 patients. Bacteriological examination and pure tone audiometry in acute otitis media are important for detecting the severity of the disease, determining the administration of steroids, and for the selection of antibiotics.

Acute Disease

[A case of Guillain-Barrè syndrome with severe sciatica preceding motor paralysis in the lower extremities].

A 62-year-old man was referred to our clinic for a three-day history of intractable sciatic pain. The pain was so severe that he could not sleep for days. Initially there was no neuronal sign except for the pain, nor any abnormality in lumbar CT and MRI. Cerebrospinal fluid sampled on the third hospital day revealed elevated protein (192 mg.dl-1) with minimal cells. On the 4th hospital day motor weakness of lower extremities appeared and he developed complete flaccid paraplegia in a week without loss of sensation. He was diagnosed as Guillain-Barrè syndrome. For the relief of this pain, intermittent injection of 4 ml 0.25% bupivacaine from lumbar epidural catheter was started on the admission day. By using patient controlled analgesia system, we could treat his repetitive demand timely and well control the pain. The leg weakness started to recover slowly from the 13th hospital day and at the same time the pain started to diminish rapidly in a few days. From the 20th hospital day he started to have rehabilitation in walking and recovered completely in two months without complication. We should remember that sudden onset of severe pain is one of early symptoms of Guillain-Barrè syndrome.

Analgesia, Epidural

Stable expression of antisense Rb-1 RNA inhibits terminal differentiation of mouse myoblast C2 cells.

To determine the roles of the retinoblastoma gene (Rb-1) in skeletal muscle differentiation in vitro, we isolated C2 myoblasts stably expressing an antisense RNA directed to the 3'-untranslated region (3'UTR) of Rb-1 mRNA. The levels of Rb-1 mRNA and its product (pRb) in the clones transfected with antisense Rb were markedly decreased to 25-35% of those in the control clone. Cell growth of the clones was accelerated, especially in medium containing low concentrations of fetal calf serum. Even in differentiation medium with a low mitogen level, the antisense Rb clones proliferated as single-nucleated myoblast-like cells without expressing the sarcometric myosin heavy chain protein, whereas the control clone formed highly multinucleated myotubes after 4 days of culture under the same conditions. Under this condition, the levels of Rb-1 mRNA and pRb in the antisense Rb clones were 30-50% of those in the control clone, and no divergent increase in the Rb-family protein p107 expression was observed. This inhibited differentiation was abrogated by reintroducing expression vectors for the sense 3'UTR of Rb-1 mRNA or Rb-1 mRNA lacking its 3'UTR to the clone transfected with antisense Rb. In the antisense Rb clone cultured in differentiation medium, the amounts of MyoD and myogenin mRNA were markedly decreased on the 2nd day of culture in the differentiation medium. The expression of cell cycle-promoting genes including E2F-1 and cyclin D1 was up-regulated throughout the experiment. These results demonstrate that pRb is essential for the completion of terminal differentiation in C2 cells.

Actins

A mutant strain of mouse FM3A cells defective in apoptotic DNA fragmentation.

A mutant strain of mouse FM3A cells was defective in apoptotic DNA fragmentation. Upon DNA damage by UV-radiation, the strain did not form DNA fragments, though it accumulated a large amount of p53 protein as did wild-type cells. No DNA fragmentation was observed when the strain was subjected to other kinds of DNA damaging agents such as X ray-radiation, or addition of etoposide to the culture medium. Other well-known biological stimulations inducing apoptosis, serum depletion or addition of TNF-alpha to the culture medium, also failed to cause DNA fragmentation in the cells. Using this apoptosis-deficient strain, we characterized another apoptotic reaction, export of phosphatidylserine to the cell surface. This molecule was exported onto the surface in both wild-type and the mutant cells when cells were treated with TNF-alpha and cycloheximide. Thus, the export of phosphatidylserine is suggested to be independent of the pathway for apoptotic DNA fragmentation.

Animals

Apoptosis was promoted at a nonpermissive temperature in DNA replication-defective temperature-sensitive mutants of mouse FM3A cells.

Apoptosis was promoted at the nonpermissive temperature in some temperature-sensitive (ts) mutant strains of mouse FM3A cells deficient in initiation of DNA replication. We examined expression of cell cycle regulation genes in the four ts mutant strains and found that two strains, tsFT107 and tsFT111, exhibited marked accumulation of p53 protein by a posttranscriptional mechanism at 16 h after temperature up-shift. These two strains also exhibited high levels of p21 mRNA expression, repression of cyclin A and D1 mRNAs, and obvious accumulation of underphosphorylated retinoblastoma protein. Only these two strains died by apoptosis at day 3 after up-shift, although no change was observed in the level of bax mRNA. These results suggest the existence of two types of responses after temperature up-shift in the four temperature-sensitive cell strains of the initiation of DNA replication: one type directs inappropriate DNA replication that then may produce endogenous DNA damage, p53-mediated cell cycle arrest, and subsequent apoptosis, while the other type exhibits only the p53-independent cell cycle arrest.

Animals

Age-dependent changes in metallothionein levels in liver and kidney of the Japanese.

Samples of liver, renal cortex, and medulla were obtained from 55 forensic autopsies (0- to 95-yr-old Japanese). Metallothionein (MT) was determined by the Ag-hem or Cd-hem method. Zinc (Zn), copper (Cu), and cadmium (Cd) were determined by atomic absorption spectrophotometry. The mean levels of MT were 250 micrograms/g in the liver, and 394 micrograms/g (cortex) and 191 micrograms/g (medulla) in the kidney. Age-dependent changes were observed in both the liver and kidney. In the liver, MT level decreased during infancy and increased thereafter with age. Similar age-dependent changes in the levels of Zn and Cu were observed. In the kidney cortex, MT level increased with age, although no correlation was found after middle age. The levels of Cd and Zn also increased with age until middle age; however, they decreased thereafter. These results suggest that age-dependent changes in renal MT levels are associated with accumulation of Cd.

Adolescent