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T Nagase

Publications and source records attributed to T Nagase.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterization of long cDNA clones from human adult spleen.

As an extension of our analysis of long cDNAs, we here report the characterization of cDNA clones from human adult spleen. From 2000 cDNA clones randomly sampled from a size-fractionated human spleen cDNA library (average size 4.5 kb), 97 clones were selected for sequencing according to their ability to code for protein at the 5'-end sequences and the novelty of their end sequences. The sequence data of these clones demonstrated that 87 out of 97 cDNA clones were derived from independent human genes. The average sizes of the inserts and corresponding open reading frames of these 87 cDNAs reached 4.5 kb and 1.4 kb (corresponding to 468 amino acid residues), respectively. In addition to these sequence analyses in silico, the expression profiles of the genes were also studied in ten human adult tissues by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay. The results indicated that spleen could be used as an additional source of human long cDNAs to complement the list of human genes.

Alternative Splicing↗
PubMed2000

The pineal gland is not essential for circadian expression of rat period homologue (rper2) mRNA in the suprachiasmatic nucleus and peripheral tissues.

To investigate the functional involvement of the pineal gland in circadian expression of the rat period homolog gene (rPer2) in the suprachiasmatic nucleus (SCN) and peripheral tissues, we performed Northern blot analysis in tissues from pinealectomized rats. The ectomy did not have any significant effects on rPer2 mRNA expression patterns both in a daily light-dark condition and in a constant darkness. These results suggest that the rhythmic secretion of pineal melatonin is not essential for the circadian expression of clock genes in the SCN and other peripheral tissues of rats.

Animals↗
PubMed2000

Stereoselective reduction of alpha-bromopenicillanates by tributylphosphine.

[reaction: see text] Diastereoselective reduction of 6-bromo-6-substituted penicillanate esters has been achieved by treatment with tributylphosphine to give 6-substituted penicillanate esters. This reaction would appear to proceed through a phosphonium beta-lactam enolate species, followed by a diastereoselective protonation. This method has the advantage of being simple to carry out and it is mild, gives high diastereoselectivity, and should tolerate a number of functional groups in the substrates. Implications of these observations are discussed.

Esters↗
PubMed2000

The yeast exchange assay, a new complementary method to screen for Dbl-like protein specificity: identification of a novel RhoA exchange factor.

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.

Actin Cytoskeleton↗
PubMed2000

Prediction of the coding sequences of unidentified human genes. XVIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In our series of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1544 to KIAA1643, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here reached 4.6 kb and 2.8 kb (930 amino acid residues), respectively. By computer-assisted database search of the deduced amino acid sequences, 48 predicted gene products were classified into the five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. Homology search against the databases for proteins deduced from yeast, nematode and fly full genome sequences revealed only one gene (KIAA1630) was entirely conserved among human and these three organisms in the 100 genes reported here. Additionally, their chromosomal loci were determined by using human-rodent hybrid panels unless they were already assigned in the public databases. Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Amino Acid Sequence↗
PubMed2000

Prediction of the coding sequences of unidentified human genes. XVII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

To provide information regarding the coding sequences of unidentified human genes, we have conducted a sequencing project of human cDNAs which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unknown human genes, named KIAA1444 to KIAA1543, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.4 kb and 2.6 kb (856 amino acid residues), respectively. Database searches of the predicted amino acid sequences classified 53 predicted gene products into the following five functional categories: cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. It was also revealed that homologues for 32 KIAA gene products were detected in the databases, which were similar in sequence through almost their entire regions. Additionally, the chromosomal loci of the genes were determined by using human-rodent hybrid panels unless their chromosomal loci were already assigned in the public databases. The expression levels of the genes were monitored in spinal cord, fetal brain and fetal liver, as well as in 10 human tissues and 8 brain regions, by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Adult↗
PubMed2000

Prediction of the coding sequences of unidentified human genes. XVI. The complete sequences of 150 new cDNA clones from brain which code for large proteins in vitro.

We have carried out a human cDNA sequencing project to accumulate information regarding the coding sequences of unidentified human genes. As an extension of the preceding reports, we herein present the entire sequences of 150 cDNA clones of unknown human genes, named KIAA1294 to KIAA1443, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here reached 4.8 kb and 2.7 kb (910 amino acid residues), respectively. From sequence similarities and protein motifs, 73 predicted gene products were functionally annotated and 97% of them were classified into the following four functional categories: cell signaling/communication, nucleic acid management, cell structure/motility and protein management. Additionally, the chromosomal loci of the genes were assigned by using human-rodent hybrid panels for those genes whose mapping data were not available in the public databases. The expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Adult↗
PubMed2000

HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge

Animals↗
PubMed2000

New EIA technique for tyrosinase in human melanocytes and its application.

The expression of tyrosinase in melanocytes relates to skin pigmentation or depigmentation. Although many types of drugs with whitening effects are well known, neither the definite effect nor the mechanism underlying the effect has been elucidated. In this study, we attempted to develop the rapid and simple EIA technique for tyrosinase protein, then this technique was applied to normal human cultured melanocytes. When primary antibody and tyrosinase were incubated in non-coated 96-well microtitre plates for 48 hours at 4 degrees C, then the solution in tyrosinase-coated plate was further incubated for another 1 hour at 37 degrees C. Thus the best results were obtained. The developed EIA system could detect authentic tyrosinase until 0.1-1.0 ng/mL. This EIA technique could also be applied to human cultured melanocytes. The melanocytes cultured with endothelin-1 induced tyrosinase like immune reactive protein. The protein induction with endothelin-1 was suppressed by BQ 123, ETa receptor antagonists. The simple EIA technique developed for tyrosinase may give a clue to determination of the onset mechanisms underlying pigmental diseases of the skin as well as the mechanisms underlying the effects of various whitening drugs.

Cells, Cultured↗
PubMed2000

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors.

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.

Animals↗
PubMed2000

Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.

Adult respiratory distress syndrome (ARDS) is characterized by acute lung injury with a high mortality rate and yet its mechanism is poorly understood. Sepsis syndrome and acid aspiration are the most frequent causes of ARDS, leading to increased lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration and respiratory failure. Using a murine model of acute lung injury induced by septic syndrome or acid aspiration, we investigated the role of cytosolic phospholipase A2 (cPLA2) in ARDS. We found that disruption of the gene encoding cPLA2 significantly reduced pulmonary edema, PMN sequestration and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. Acute lung injury induced by acid aspiration was similarly reduced in mice with a disrupted cpla2 gene. Our observations suggest that cPLA2 is a mediator of acute lung injury induced by sepsis syndrome or acid aspiration. Thus, the inhibition of cPLA2-initiated pathways may provide a therapeutic approach to acute lung injury, for which no pharmaceutical agents are currently effective.

Animals↗
PubMed2000

NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain.

The BRCT (BRCA1 C-terminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control. The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089). This gene product has been described as one of the BRCT superfamily proteins. However, its biological significance has been unclarified. Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal. In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity. The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells. Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation. These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Detection of spurious interruptions of protein-coding regions in cloned cDNA sequences by GeneMark analysis.

cDNA is an artificial copy of mRNA and, therefore, no cDNA can be completely free from suspicion of cloning errors. Because overlooking these cloning errors results in serious misinterpretation of cDNA sequences, development of an alerting system targeting spurious sequences in cloned cDNAs is an urgent requirement for massive cDNA sequence analysis. We describe here the application of a modified GeneMark program, originally designed for prokaryotic gene finding, for detection of artifacts in cDNA clones. This program serves to provide a warning when any spurious split of protein-coding regions is detected through statistical analysis of cDNA sequences based on Markov models. In this study, 817 cDNA sequences deposited in public databases by us were subjected to analysis using this alerting system to assess its sensitivity and specificity. The results indicated that any spurious split of protein-coding regions in cloned cDNAs could be sensitively detected and systematically revised by means of this system after the experimental validation of the alerts. Furthermore, this study offered us, for the first time, statistical data regarding the rates and types of errors causing protein-coding splits in cloned cDNAs obtained by conventional cloning methods.

Alternative Splicing↗
PubMed2000

Effect of ions on antibacterial activity of human beta defensin 2.

Human beta defensin 2 (HBD-2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD-2. This activity, measured in 10 mM phosphate buffer at a concentration of 20 microg/ml, reduced significantly in the presence of 100 and 150 mM sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10 mM of Tris buffer significantly inactivated HBD-2 at much lower concentrations (more than or equal to 0.01 mM and 0.05 mM, respectively) than the monovalent ions did. These findings suggest that HBD-2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.

Anti-Bacterial Agents↗
PubMed2000

Comparison of hypocalcemic hypercalciuria between patients with idiopathic hypoparathyroidism and those with gain-of-function mutations in the calcium-sensing receptor: is it possible to differentiate the two disorders?

Gain-of-function mutations in the calcium ion-sensing receptor (CaR) cause hypocalcemia with low PTH levels. It is stated that patients with activating CaR mutations generally show milder degree of hypocalcemia before treatment and more profound hypercalciuria during treatment than those with idiopathic hypoparathyroidism (IHP). To test this validity we analyzed the data of serum and urinary calcium collected from 85 patients with IHP and 15 with activating CaR mutations. The mean (+/-SEM) serum calcium concentration before treatment was significantly higher (P: < 0.001) in patients with activating CaR mutations (1.76 +/- 0.05 mmol/L; n = 15) than in those with IHP (1.41 +/- 0.03; n = 58), but there was a substantial overlap in the range of hypocalcemia between the two groups (1.25-2. 05 vs. 0.90-1.95). The mean urinary calcium/creatinine ratio (Ca/Cr) in patients with activating CaR mutations before treatment (0.362 +/- 0.045 mmol/mmol; n = 9) was almost equal to that in normocalcemic controls (0.331 +/- 0.022; n = 56) and markedly higher (P: < 0.001) than in patients with IHP (0.093 +/- 0.008; n = 57). The overlap of pretreatment urinary Ca/Cr between the 2 disorders was relatively small; subnormal urinary Ca/Cr was observed in only 1 of 9 patients with CaR mutations and in the majority (49 of 57) of patients with IHP. In contrast to pretreatment findings, the degree of hypercalciuria during treatment was not different between the 2 disorders. The data points of urinary Ca/Cr plotted as a function of the serum calcium concentration were not separable between patients with CaR mutations (n = 8) and those with IHP (n = 40). Comparison of urinary Ca/Cr between 2 patients with a CaR mutation and 7 with IHP over a wide range of serum calcium concentrations measured during 4-8 yr of treatment also indicated that the 2 disorders were inseparable. These results suggested that inappropriately normal urinary Ca/Cr in patients with untreated hypocalcemia, mostly of mild degree, might be a better biochemical clue than the development of severe hypercalciuria during treatment to suspect gain-of-function mutations in the CaR.

Adolescent↗
PubMed2000

Crouzon syndrome with acanthosis nigricans: case report and mutational analysis.

OBJECTIVE: To describe the 22nd case of Crouzan syndrome with acanthosis nigricans, a hyperkeratotic skin disorder with hyperpigmentation. METHODS: DNA analysis and sequencing of the FGFR3 gene were performed. RESULTS: The 13-year-old Japanese boy described here also had dyspnea, facial palsy, sensorineural hearing loss, and skeletal and mental retardation. Examination of a skin biopsy specimen revealed the typical findings of acanthosis nigricans. Genetic analysis revealed the Ala391Glu mutation in one FGFR3 gene. CONCLUSIONS: Crouzon syndrome with acanthosis nigricans is a distinct clinical entity different from classic Crouzon syndrome.

Acanthosis Nigricans↗
PubMed2000

[Sleep apnea syndrome and hypertension using desaturation index].

To examine the roles of obstructive apnea (OA) and central apnea (CA) in oxygen desaturation on hypertension and sleep apnea syndrome (SAS), we performed a sleep study on 41 elderly subjects (mean age 69.5 +/- 6.8 years, male:female = 31:10). Nocturnal oxygen desaturation was documented with a pulse oximeter and apneas (OA and CA) were diagnosed on the basis of results of respiratory inductive plethysmography and oronasal flow. Significant desaturation (SDS, greater than 5% drop in SpO2 from baseline value) and desaturation index (DI; epsilon SDS (%) x duration (hour)) were calculated using the continuous nocturnal monitoring system with a pulse oximeter. We defined central type apnea above 50% as the central type group (n = 8, mean age 58.6 +/- 2.9, mean BMI 21.3 +/- 1.0, male:female = 7:1), and obstructive type and mixed type apnea above 50% as the obstructive type group (n = 21, mean age 70.0 +/- 3.2, mean BMI 25.3 +/- 1.0, male:female = 17:4). Other subjects were assigned to the control group (n = 12, mean age 64.3 +/- 2.3, mean BMI 23.8 +/- 1.2, male:female = 7:5). The DI (delta 5%) of the central type was 0.34 +/- 0.17, and that of the obstructive type was 1.78 +/- 0.7 showing a significant increase in the latter compared to the control group (p < 0.02). The DI (< 90%) of the central type was 0.14 +/- 0.07, and that of the obstructive type was 1.72 +/- 0.75, and that of the obstructive type was significantly greater than in the control group (p < 0.05) and central type (p < 0.05). There were 4 cases (33.3%) with hypertension in the control group and 4 cases (50.0%) with hypertension in the central type group, but there were 15 cases (71.4%) with hypertension in the obstructive type group. Hypertensive prevalence in the obstructive group was significantly more than in the control group (p < 0.05). No significant difference in body mass index or age were seen in the obstructive group and control group. There was a significant correlation between mean blood pressure and apnea index (AI). The AI of the hypertensive group was significantly higher than that of the normotensive group (p < 0.001). These results suggest that subjects with significant obstructive apneas may be at greater risk for hypertension than subjects with central apneas and that hypertension in the pathogenesis of SAS may be related to the severity of apneas rather than oxyhemoglobin desaturation.

Adult↗