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Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 127 records · Page 7Linked to original sources
PubMed1998

Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins.

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.

Adenoviridae↗
PubMed1998

Molecular cloning and characterization of a novel nuclear protein kinase in mice.

We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers. The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase. The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s). This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25. The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei. Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver. These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of p56dok-2 defines a new family of RasGAP-binding proteins.

Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210(bcr-abl), a chimeric protein with tyrosine kinase activity. Substrates for p210(bcr-abl) are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56(dok-2) (Dok-2), from p210(bcr-abl) expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120(RasGAP). Dok-2 was shown to be 35% identical to p62(dok-1), a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210(bcr-abl) in CML.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase.

BACKGROUND: In eukaryotic cells, newly synthesized histone H4 is acetylated at lysines 5 and 12, a transient modification erased by deacetylases shortly after deposition of histones into chromosomes. Genetic studies in Saccharomyces cerevisiae revealed that acetylation of newly synthesized histones H3 and H4 is likely to be important for maintaining cell viability; the precise biochemical function of this acetylation is not known, however. The identification of enzymes mediating site-specific acetylation of H4 at Lys5 and Lys12 may help explain the function of the acetylation of newly synthesized histones. RESULTS: A cDNA encoding the catalytic subunit of the human Hat1 acetyltransferase was cloned and, using specific antibodies, the Hat1 holoenzyme was purified from human 293 cells. The human enzyme acetylates soluble but not nucleosomal H4 at Lys5 and Lys12 and acetylates histone H2A at Lys5. Unexpectedly, we found Hat1 in the nucleus of S-phase cells. Like its yeast counterpart, the human holoenzyme consists of two subunits: a catalytic subunit, Hat1, and a subunit that binds core histones, p46, which greatly stimulates the acetyltransferase activity of Hat1. Both p46 and the highly related p48 polypeptide (the small subunit of human chromatin assembly factor 1; CAF-1) bind directly to helix 1 of histone H4, a region that is not accessible when H4 is in chromatin. CONCLUSIONS: We suggest that p46 and p48 are core-histone-binding subunits that target chromatin assembly factors, chromatin remodeling factors, histone acetyltransferases and histone deacetylases to their histone substrates in a manner that is regulated by nucleosomal DNA.

Acetylation↗
PubMed1998

[A case of the pulmonary thrombosis caused by unilateral pulmonary artery occulusion test].

A 71-year-old male, diagnosed as lung cancer, underwent unilateral pulmonary occulusion test. Through the guidewire, 7.5 Fr thermodilution catheter with occlusion balloon was introduced to the left pulmonary artery from the right internal jugular vein. Heparinized physiological saline solution was injected into the distal site of the occulusion. The occulusion time was 15 minutes. Pulmonary artery pressure and wedge pressure were within normal range. Soon after the examination, the pulmonary arteriogram (PAG) showed the defect of the branch to the lingular segment and the lower lobe. We made a diagnosis of pulmonary thrombosis. Three days after the administration of urokinase and heparin, both pulmonary perfusion scintigram and PAG exhibited the reperfusion to these areas. After the thrombolytic therapy was accomplished, antithrombin III and protein C in the serum showed within normal range. It was possible that the damage on the intima due to the thermodilution catheter or the guidewire and the following blood congestion by the pulmonary artery occulusion caused the thrombosis.

Aged↗
PubMed1998

Serum surfactant protein A levels in healthy individuals are increased in smokers.

Serum levels of surfactant protein A (SP-A) were studied in 237 healthy subjects in relation to sex, age, and smoking habits. SP-A values in male smokers were significantly higher than those in male nonsmokers (p < 0.001). The amount of cigarette smoking did not correlate significantly with SP-A values, however. SP-A values in young nonsmoking males and females were somewhat lower than those in older, but without significant difference. No significant difference in values was found between the sexes. We conclude that (1) smoking increases serum levels of SP-A, and (2) SP-A serum levels are not affected by age and sex.

Adolescent↗
PubMed1998

Involvement of blood-group-B-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula.

Despite their wide distribution in various organisms, no physiological roles have been proposed for the human blood-group-ABO (ABH)-active trisaccharides. Here we show that monoclonal antibodies against human blood-group-B-active trisaccharides (B-substance) completely block the Ca2+-dependent cell-cell adhesion system of frog (Xenopus laevis) embryonic cells. Synthetic B-substance or B-active glycopeptides also disrupt the Ca2+ -dependent cell-cell adhesion. These results suggest that blood-group-B-active substances play a role in cell-cell adhesion. Blood-group-B-active substances were found as glycoproteins and as glycosphingolipids. In order to identify B-active glycoproteins active in cell-cell adhesion, we purified B-active membrane glycoproteins by two-dimensional electrophoresis and found that they are 45- to 58-kDa proteins with pI(s) ranging from 4.0 to 5.3. They are glycosylphosphatidyl inositol (GPI) anchored. Amino acid sequence analysis showed that the purified B-active GPI-anchored proteins are homologues of soluble Xenopus cortical granule lectins (CGL). The results suggest that the B-active membrane glycoproteins are GPI-anchored forms of the lectin and are directly involved in frog Ca 2+-dependent cell-cell adhesion.

ABO Blood-Group System↗
PubMed1998

Limited axillary thoracotomy vs video-assisted thoracoscopic surgery for spontaneous pneumothorax.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) has been reported to have a higher pneumothorax recurrence rate than limited axillary thoracotomy (LAT). We investigated the cause of pneumothorax recurrence after VATS by comparing surgical results for VATS and LAT. METHODS: Ninety-five patients with spontaneous pneumothorax underwent resection of pulmonary bullae by VATS (n = 51) or LAT (n = 44). Operating duration, bleeding during surgery, number of resected bullae, duration of postoperative chest tube drainage, postoperative hospital stay, postoperative complication, and pneumothorax recurrence were analyzed to compare VATS and LAT in a retrospective study. RESULTS: The duration of surgery, postoperative chest tube drainage, and postoperative hospital stay was significantly shorter in VATS than in LAT cases (p < 0.0005 and p < 0.005). Bleeding during surgery was significantly less in VATS than in LAT cases (p < 0.005). Numbers of resected bullae were significantly lower in VATS (2.7 +/- 2.1) than in LAT cases (3.9 +/- 2.7) (p < 0.05). Postoperative pneumothorax recurrence was more frequent in VATS (13.7%) than in LAT cases (6.8%), but there was no significant difference. CONCLUSIONS: VATS has many advantages over LAT in treating spontaneous pneumothorax, although the pneumothorax recurrence rate in VATS cases was double that in LAT cases. The lower number of resected bullae in VATS than in LAT cases suggests that overlooking bullae in operation could be responsible for the high recurrence rate in VATS cases. We recommend additional pleurodesis in VATS for spontaneous pneumothorax to prevent postoperative pneumothorax recurrence.

Adolescent↗
PubMed1998

Detection of Epstein-Barr virus infection in the epithelial cells and lymphocytes of non-neoplastic tonsils by in situ hybridization and in situ PCR.

Non-neoplastic tonsils were analyzed for detection of Epstein-Barr virus (EBV)-positive cells by in situ hybridization and in situ PCR. EBV-encoded small nuclear RNA 1(EBER1)-positive cells were found in 28.2% of the tonsils and were evenly localized in the extrafollicular area and within germinal centers. Latent membrane protein 1 (LMP1)-positive cells were also dispersed in the extrafollicular and germinal center. Using in situ DNA-DNA hybridization, the EBV-positive signals were observed in the upper epithelial cell layers of the tonsils. In addition, in situ PCR detected EBV DNA-positive cells in the lower epithelial cell layers and lymphoid cells.

Chronic Disease↗
PubMed1998

The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points.

We are using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. Previous studies demonstrated that the use of the muscle-specific exon is associated with the use of distant branch points located 147-153 nt upstream of the 3' splice site. In addition, at least one protein, the polypyrimidine tract binding protein (PTB), specifically interacts with critical cis-acting sequences upstream of exon 7 that are involved in blocking the use of this alternative exon in nonmuscle cells. In order to further study the role of PTB, monoclonal antibodies to PTB were prepared. Anti-PTB antibodies did not inhibit the binding of PTB to RNA because they were able to supershift RNA-PTB complexes. To determine if additional proteins interact with sequences within the pre-mRNA, 35S-met-labeled nuclear extracts from HeLa cells were mixed with RNAs and the RNA-protein complexes were recovered by immunoprecipitation using antibodies to PTB. When RNAs containing intron 6 were added to an 35S-met-labeled nuclear extract, precipitation with PTB antibodies showed a novel set of proteins. By contrast, addition of RNAs containing introns 5 or 7 gave the same results as no RNA, indicating that these RNAs are unable to form stable complexes with PTB. These results are in agreement with our previous studies demonstrating that PTB interacts with sequences within intron 6, but not with sequences within introns 5 and 7. When 35S-met-labeled HeLa nuclear extracts were mixed with biotinylated RNA containing intron 6 and the RNA-protein complexes were recovered using streptavidin-agarose beads, an identical pattern of proteins was observed when compared with the immunoprecipitation assay. Analysis of the proteins that assembled on introns 5, 6, or 7 using biotinylated RNA revealed a unique set of proteins that interact with each of these sequences. The composition of proteins interacting with sequences associated with the use of the 3' splice site of intron 6 included proteins of 30, 40, 55, 60, 65, 70, 80, and 100 kDa. Microsequencing identified two of the proteins to be Sam68 and the Far Upstream Element Binding Protein (FBP) from the c-myc gene. In addition, a comparison of the proteins that assemble on introns from the alpha- and beta-TM genes that utilize distant branch points revealed common as well as unique proteins that assemble on these introns. These studies identify a set of proteins, in addition to PTB, that are likely involved in the use of distant branch sites associated with the use of alternatively spliced introns.

Alternative Splicing↗
PubMed1998

The snRNP-free U1A (SF-A) complex(es): identification of the largest subunit as PSF, the polypyrimidine-tract binding protein-associated splicing factor.

We have previously shown that a specific monoclonal antibody prepared against the U1A protein, MAb 12E12, is unique in its ability to recognize a form of U1A which is not associated with the U1snRNP. This unique form of U1A, termed snRNP-free U1A or SF-A, was found to be complexed with a novel set of non-snRNP proteins (O'Connor et al., 1997, RNA 3:1444-1455). Here we demonstrate that the largest protein in these SF-A complex(es), p105, is the polypyrimidine-tract binding protein-associated factor (PSF), an auxiliary splicing factor. We show that PSF copurifies and co-immunoprecipitates with SF-A from 293T cell nucleoplasm and that it interacts with SF-A in vitro. In addition, we show that MAb 12E12 inhibits both splicing and polyadenylation in an in vitro coupled splicing and polyadenylation reaction. This suggests that SF-A and/or the SF-A complex(es) perform an important function in both processing reactions and possibly in last exon definition.

Amino Acid Sequence↗
PubMed1998

Replication focus-forming activity 1 and the Werner syndrome gene product.

The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.

Adenosine Triphosphate↗
PubMed1998

Electroencephalogram abnormality and high-dose busulfan in conditioning regimens for stem cell transplantation.

High-dose busulfan (BU) is widely used in combined chemotherapy before allogeneic or autologous bone marrow transplantation. Convulsions are reported as a side-effect of high-dose BU. We recorded electroencephalograms (EEGs) before and on the third day of BU administration in 22 patients. Abnormal EEGs were observed on the third day in 13 cases (59%). These patients were older (P < 0.05) and had had larger doses of BU (P < 0.025) than the nine patients with normal EEGs. Convulsions occurred in two of the 22 patients, one of whom was receiving prophylaxis with phenytoin. Gamma aminobutyric acid (GABA), a natural mediator of defense against epileptic activity, concentrations in cerebrospinal fluid measured before and after administration of BU showed no definite changes.

Adolescent↗
PubMed1998

Behavioral characteristics of 187 young adults with autism.

A survey was conducted on the present behavioral characteristics of 187 cases of adult autism in patients over 18 years of age employing Achenbach's Child Behavior Checklist (CBCL). When their behavioral characteristics were evaluated in relation to Present Language Developmental Level (PLDL) and Present Adaptive Level (PAL), it was seen that greater variation in behavior characteristics was seen among those exhibiting increasingly lower PLDL and PAL scores. Behavior characteristics reminiscent of depression were noted even among those exhibiting high PLDL. Behavior pointing to obsession was found in common among almost all cases of autism irrespective of PLDL or PAL. Psychotic symptoms such as hallucinations and delusions were absent in most cases. The results of the present study were indicative not only of the significance of obsessive behavior in autism, but also its significance in terms of delving further into the psychopathology of the disorder.

Adolescent↗
PubMed1998

Perception metamorphosis phenomenon in autism.

It is well known that various perceptual abnormalities exist in autism. However, because perceptual phenomena are intersubjective, a phenomenological approach is required for getting hold of the reality of the modes of perception involved in autism. From this standpoint, the author has proposed the concept of 'perception metamorphosis phenomenon' (PMP) as the mode of perception peculiar to autistics. This mode of perception is notable to some degree in infancy and adolescence, and points to the appearance of behavior that is indicative of the environmental world being perceived in a manner different from before by the autistic child. The phenomenon has been classified into three basic categories according to the aspect of perception: (i) visual PMP; (ii) auditory PMP; and (iii) situational PMP. The proposal of this concept was made with the objective of capturing the onset of autism or the mechanism of appearance of the various symptoms from a more phenomenological viewpoint, to serve as a possible starting point for understanding the inner world of autistics. The proposal was made emphasizing the validity of this approach in mapping out new therapeutic approaches and for re-investigating the relationship between autism and schizophrenia.

Adolescent↗
PubMed1998

Parvovirus B19-induced anemia as the presenting manifestation of X-linked hyper-IgM syndrome.

Parvovirus B19 (B19) can cause chronic anemia due to persistent infection in immunocompromised hosts who cannot produce neutralizing antibody necessary for clearing B19. Three patients with X-linked hyper-IgM syndrome (XHIM), who were all asymptomatic until they developed B19-induced chronic anemia at the ages of 8, 14, and 17 years, respectively, were found to have mutations of the CD40L gene, including a missense mutation (T254M), a nonsense mutation resulting in a new initiation codon and loss of the intracellular domain (R11X), and a splice site mutation (nt 309+2t-->a). Antibody responses to the T cell-dependent antigen, bacteriophage phiX174, were impaired, but neutralizing antibody titers were higher than in XHIM patients with classic phenotype. All 3 patients responded to intravenous immune globulin (IVIG) treatment. Certain mutations of the CD40L gene result in a mild XHIM phenotype that may become apparent following B19 infection in patients not on IVIG therapy and therefore not protected from B19 infection.

Adolescent↗
PubMed1998

Gender differences in self-evaluated sleep quality and activity of middle-aged and aged subjects.

In order to investigate the gender difference of sleep and activity in middle-aged and aged individuals, home-based sleep was self-evaluated for sleep quality and activity for 5 nights in 20 healthy adults (50-76 years old; 11 women, nine men). There was no significant gender difference for subjective sleep quality. However, the activity level and movement index at night were significantly higher in men than in women, and the activity level during the day was significantly lower in men than in women. The objective sleep quality of men was significantly worse than that of women, however, subjective sleep quality does not differ.

Activity Cycles↗