[Studies of regulation of gene expression developed from genomic science].
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Animals↗
Biomedical subjects
T Nagase
Publications and source records attributed to T Nagase.
Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain.
We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5'-end single-pass sequence data and the GeneMark program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5'-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted.
5' Untranslated Regions↗
Prediction of the coding sequences of unidentified human genes. XV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.
In order to obtain information on the coding sequences of unidentified human genes, we newly determined the sequences of 100 cDNA clones of unknown human genes, which we named KIAA1193 to KIAA1292, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The results of our particular strategy to select cDNA clones which have the potentiality of coding for large proteins in vitro revealed that the average sizes of the inserts and the corresponding open reading frames reached 5.2 kb and 2.8 kb (933 amino acid residues), respectively. By the computational analysis of the predicted amino acid sequences against the OWL and Pfam databases, 58 predicted gene products were classified into the following five functional categories: cell signaling/communication, cell structure/motility, nucleic acid management, protein management and metabolism. It was also found that 30 gene products had homologues in the public databases which were similar in sequence throughout almost their entire regions to the newly identified genes. The chromosomal loci of the genes were assigned by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of the genes were 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↗
Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.
To extend our cDNA project for accumulating basic information on unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human adult and fetal brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA1019 to KIAA1118. The sequencing of these clones revealed that the average size of the inserts and corresponding open reading frames were 5.0 kb and 2.6 kb (880 amino acid residues), respectively. Database search of the predicted amino acid sequences classified 58 predicted gene products into the five functional categories, such as cell signaling/communication, cell structure/motility, nucleic acid management, protein management and cell division. It was also found that, for 34 gene products, homologues were detected in the databases, which were similar in sequence through almost the entire regions. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Adult↗
Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.
We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.
Amino Acid Sequence↗
A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p.
Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1). Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases. The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues. The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495-967) with homology to HDA1p. In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex.
Amino Acid Sequence↗
Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.
A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.
Apoenzymes↗
The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?
Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.
Alpha-Globulins↗
Prediction of the coding sequences of unidentified human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.
As a part of our cDNA project for deducing the coding sequence of unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0919 to KIAA1018. The sequencing of these clones revealed that the average sizes of the inserts and corresponding open reading frames were 4.9 kb and 2.6 kb (882 amino acid residues), respectively. A computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes contained sequences similar to known genes, 53% of which (46 genes) were categorized as proteins relating to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Animals↗
Effect of farnesyltransferase overexpression on cell growth and transformation.
A series of studies using farnesyltransferase (FTase) inhibitors that the inhibition of FTase function suppresses the growth of ras-transformed cells in vitro and in vivo. However, whether FTase is directly involved in the regulation of cell proliferation remains to be demonstrated. To investigate whether overexpression of FTase results in altered cell growth and transformation, we thus used NIH3T3 cells transfected with cDNA constructs of both alpha and beta subunits of human FTase. FTase-overexpressing cells resulted in a 3- to 13-fold increase in the expression of the alpha and beta subunit protein of FTase and a 1.5- to 3-fold increase in the level of the enzyme activity compared with untransfected NIH3T3 cells or vector-transfected cells. Further investigations using metabolic labeling indicated that farnesylation of Ras was enhanced in FTase-overexpressing cells. Insulin-like growth factor-I, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) more potently enhanced DNA synthesis and anchorage-dependent growth in FTase-overexpressing cells than in control cells, in a dose-dependent manner. In particular, PDGF and bFGF also induced dose-dependently enhanced colony formation in soft agar in FTase-overexpressing cells. Furthermore, in FTase-transfectants, bFGF stimulated high activation of mitogen-activated protein kinase. Interestingly, FTase-transfectants developed progressive tumors in nude mice. Light and electron microscopy showed that the tumors were characteristic of fibrosarcoma, which were distinct from v-ras-induced tumors. Overexpression of FTase in NIH3T3 cells thus amplifies growth-factor-mediated cell growth and transformation, and FTase-overexpressing cells form tumors in nude mice.
3T3 Cells↗
HUGE: a database for human large proteins identified by Kazusa cDNA sequencing project.
HUGE is a database for human large proteins newly identified by Kazusa cDNA project, which aims to predict protein primary structures from sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are current targets of the project. More than 700 sequences of human cDNAs (average size, 5.1 kb) have been determined to date and deposited in the public databases. Notable information implied from the cDNAs and the predicted protein sequences can be obtained through HUGE via the World Wide Web at URL http://www.kazusa.or.jp/huge
Cloning, Molecular↗
Familial paroxysmal dystonic choreoathetosis: clinical findings in a large Japanese family and genetic linkage to 2q.
BACKGROUND: Paroxysmal dystonic choreoathetosis (PDC) is a rare familial movement disorder that has been mapped to chromosome 2q31-36. OBJECTIVE: To study the first Japanese family with PDC clinically and genetically. PATIENTS AND METHODS: We studied a large Japanese family in which at least 17 members in 6 generations have been affected by PDC. We interviewed and examined 26 family members, 8 of whom revealed choreoathetosis-like and dystonialike involuntary movement and 1 of whom revealed no involuntary movement but only muscle stiffness such as the aura of paroxysmal dystonic choreoathetosis (PDC). Genetic linkage studies of this family were carried out with polymorphic DNA markers. RESULTS: The attacks of involuntary movement or muscle stiffness were precipitated by ovulation, menstruation, emotional stress, or caffeine or alcohol ingestion. Magnetic resonance imaging of the brain revealed no abnormalities. Clonazepam therapy was effective for reducing the attacks, and ingestion of garlic was believed by patients to be effective for softening the attacks. An affected woman with only muscle stiffness showed remission after hysterectomy for hysteromyoma. This woman also had the disease haplotype and transferred it to her typical PDC-affected daughter. Maximal pairwise logarithm of odds scores exceeding 2.00 were obtained at D2S2250, D2S1242, D2S377, D2S2148, and D2S126. The PDC gene was demonstrated by linkage analyses to be located in a 15.3-centimorgan interval lying between D2S371 and D2S339 based on pairwise and multipoint logarithm of odds scores and obligate recombination events in affected individuals. CONCLUSIONS: Linkage of PDC to chromosome 2q32-36 was confirmed in a Japanese family. The clinical characterizations of this family with PDC include that ovulation seems also to be a precipitating factor of the attacks and that hysterectomy seems to be effective for softening the attacks. Although low-dose clonazepam treatment was most effective, garlic use was believed by affected members to be effective for softening the attacks. Furthermore, based on the results of clinical and genetic analyses, we suggest that muscle stiffness without involuntary movement may represent a forme fruste of PDC.
Athetosis↗
Circulating levels of interleukin-6, its soluble receptor and interleukin-6/interleukin-6 receptor complexes in patients with type 2 diabetes mellitus.
We evaluated the relationship between plasma fibrinogen concentration and the serum levels of interleukin-6 (IL-6), its soluble receptor, and their complex in patients with type 2 diabetes mellitus. The study comprised 57 patients with type 2 diabetes and 15 normal healthy controls. Serum levels of IL-6, soluble IL-6 receptor (IL-6R), and circulating IL-6/IL-6R complex were determined by enzyme-linked immunosorbent assays. Correlations between the different study parameters and serum IL-6, IL-6R, or IL-6/IL-6R complex levels were determined by multiple linear regression analysis. Any association between the different study parameters and the serum levels of IL-6, IL-6R, or IL-6/IL-6R complex were determined by stepwise linear regression analysis. The serum IL-6 level in diabetic subjects was significantly higher than in normal healthy controls (3.48 +/- 3.29 pg/ml vs 0.784 +/- 0.90 pg/ml, mean +/- SD, respectively, P = 0.0001). The specific optical density of the serum IL-6/IL-6R complex in diabetic patients was also significantly higher than in normal healthy controls, although there was no significant difference in the serum IL-6R level between diabetic patients and controls. The serum IL-6 concentration was correlated significantly with the HbA(1C) level (beta = 0.58, P = 0. 04) by multiple regression analysis. Stepwise regression analysis revealed that the levels of serum IL-6 (F = 8.251), HbA(1C) (F = 1. 08), and serum urea nitrogen (F = 5.603) were associated with the plasma fibrino gen concentration. These results suggest that hyperglycaemia and increased levels of serum IL-6 can increase the plasma fibrinogen concentration, one of the known risk factors for atherosclerosis in patients with type 2 diabetes mellitus.
Adult↗
Increased levels of circulating ICAM-1, VCAM-1, and L-selectin in obstructive sleep apnea syndrome.
Obstructive sleep apnea syndrome (OSAS) may be one of the most important risk factors of cardiovascular disorders, although the exact mechanism remains to be elucidated. In the present study, we hypothesized that OSAS-induced hypoxic stress might be involved in the etiology of cardiovascular disorders by activating adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and L-selectin. To examine this hypothesis, we measured circulating ICAM-1, VCAM-1, and L-selectin levels before and after sleep in OSAS patients and age-matched controls. The circulating ICAM-1, VCAM-1, and L-selectin levels increased in the OSAS patients before sleep compared with the normal subjects (ICAM-1: 392.9 +/- 48.5 vs. 201.2 +/- 55.0 ng/ml, P < 0.05; VCAM-1: 811.0 +/- 87.8 vs. 574.2 +/- 42.7 ng/ml, P < 0.05; L-selectin: 1,386.6 +/- 77.9 vs. 1,038.8 +/- 78.6 ng/ml, P < 0.01, respectively). After sleep, significantly greater levels of ICAM-1 and L-selectin, but not VCAM-1, were observed in the OSAS group. These observations suggest that OSAS-induced hypoxia activates adhesion molecules, resulting in the important risk factor of cardiovascular disorders. Treatment of OSAS can be, therefore, a potential approach to prevention of cardiovascular events.
Adult↗
Disruption of ET-1 gene enhances pulmonary responses to methacholine via functional mechanism in knockout mice.
Endothelin (ET)-1 has been shown to have various pathophysiological roles in the lung. Recently, it has been reported that ET-1 and a gene encoding ET-1 (Edn1) might be involved in airway hyperresponsiveness, which is a major feature of bronchial asthma. Meanwhile, it remains unclear whether ET-1 might be involved in airway remodeling in vivo. In the present study, we hypothesized whether ET-1 might play a role in airway remodeling, leading to altered responsiveness. To test this hypothesis, we investigated airway function in vivo and airway wall structure in Edn1(+/-) heterozygous knockout mice, which genetically produce lower levels of ET-1, and Edn1(+/+) wild-type mice. In the physiological study, enhanced responses in lung elastance and resistance to methacholine administration were observed in Edn1(+/-) mice, whereas there was no difference in serotonin responsiveness. In the morphometric study, there were no differences in either lamina propria or airway smooth muscle thickness between Edn1(+/-) mice and Edn1(+/+) mice. These findings suggest that ET-1 gene disruption is involved in methacholine pulmonary hyperresponsiveness via functional mechanism, but not airway remodeling, in mice. The ET-1 knockout mice may provide appropriate models to study diseases related to ET-1 metabolism.
Airway Resistance↗
Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice.
Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration-induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration-induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.
Animals↗
Recombinant E1-deleted adenoviral vectors induce apoptosis in a rat airway epithelial mucous goblet cell line.
Replication-defective adenoviruses (Ad) are used as vectors for delivering therapeutic genes to human airway cells. We examined whether E1-deleted Ad vectors (Ad5-CMV-LacZ) had effects on cell kinetics in SPOC1 cells, which is a rat airway epithelial mucous goblet cell line. There was a vector multiplicity of infection (moi)-dependent increase of the transduction efficiency of the LacZ reporter gene in SPOC cells. Cell proliferation was inhibited in the vector-infected cells compared with that in vehicle-exposed cells. However, increased cell death was observed in the vector-infected cells with a higher moi. The morphology of vector-exposed cells revealed apoptotic features including nuclear condensation and a fragmented nucleus. These results indicate that higher moi of vectors allows the cells to achieve higher gene transfer, but also induce apoptosis of infected cells. Minimizing the induction of apoptosis of vector-infected cells may be an important strategy for the prolongation of transduction efficiency of Ad vectors in airway epithelial mucous goblet cells.
Adenoviridae↗
[Adverse drug reaction in the elderly].
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Aged↗