Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 91 records · Page 5Linked to original sources

PubMed1999

atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes.

SR proteins are nuclear phosphoproteins with a characteristic Ser/Arg-rich domain and one or two RNA recognition motifs. They are highly conserved in animals and plants and play important roles in spliceosome assembly and alternative splicing regulation. We have now isolated and partially sequenced a plant protein, which crossreacts with antibodies to human SR proteins. The sequence of the corresponding cDNA and genomic clones from Arabidopsis revealed a protein, atSRp30, with strong similarity to the human SR protein SF2/ASF and to atSRp34/SR1, a previously identified SR protein, indicating that plants possess two SF2/ASF-like proteins. atSRp30 expresses alternatively spliced mRNA isoforms that are expressed differentially in various organs and during development. Overexpression of atSRp30 via a strong constitutive promoter resulted in changes in alternative splicing of several endogenous plant genes, including atSRp30 itself. Interestingly, atSRp30 overexpression resulted in a pronounced down-regulation of endogenous mRNA encoding full-length atSRp34/SR1 protein. Transgenic plants overexpressing atSRp30 showed morphological and developmental changes affecting mostly developmental phase transitions. atSRp30- and atSRp34/SR1-promoter-GUS constructs exhibited complementary expression patterns during early seedling development and root formation, with overlapping expression in floral tissues. The results of the structural and expression analyses of both genes suggest that atSRp34/SR1 acts as a general splicing factor, whereas atSRp30 functions as a specific splicing modulator.

Alternative Splicing↗

PubMed1999

Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family.

To investigate the roles of calcium-binding proteins in degranulation, we used three anti-allergic drugs, amlexanox, cromolyn and tranilast, which inhibit IgE-mediated degranulation of mast cells, as molecular probes in affinity chromatography. All of these drugs, which have different structures but similar function, scarcely bound to calmodulin in bovine lung extract, but bound to the same kinds of calcium-binding proteins, such as the 10-kDa proteins isolated in this study, calcyphosine and annexins I-V. The 10-kDa proteins obtained on three drug-coupled resins and on phenyl-Sepharose were analysed by reversed-phase HPLC. It was found that two characteristic 10-kDa proteins, one polar and one less polar, were bound with all three drugs, although S100A2 (S100L), of the S100 family, was bound with phenyl-Sepharose. The cDNA and deduced amino acid sequence proved our major polar protein to be identical with the calcium-binding protein in bovine amniotic fluid (CAAF1, S100A12). The cDNA and deduced amino acid sequence of the less-polar protein shared 95% homology with human and mouse S100A13. In addition, it was demonstrated that the native S100A12 and recombinant S100A12 and S100A13 bind to immobilized amlexanox. On the basis of these findings, we speculate that the three anti-allergic drugs might inhibit degranulation by binding with S100A12 and S100A13.

Amino Acid Sequence↗

PubMed1999

FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

Amino Acid Sequence↗

PubMed1999

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene.

In the rat beta-tropomyosin (beta-TM) gene, exons 6 and 7 are spliced alternatively in a mutually exclusive manner. Exon 6 is included in mRNA encoding nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle beta-TM. Previously, we demonstrated that a six nucleotide mutation at the 5' end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupting a putative secondary structure. The sequence in exon 7 acts as a bona fide exonic splicing silencer (ESS), which is bound specifically by a trans-acting factor. Isolation and peptide sequencing reveal that this factor is hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Binding of hnRNP H correlates with the ESS activity. Furthermore, addition of antibodies that specifically recognizes hnRNP H to the splicing reactions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP H. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue- and developmental-specific alternative splicing.

Alternative Splicing↗

PubMed1999

The type 2C Ser/Thr phosphatase PP2Cgamma is a pre-mRNA splicing factor.

To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions. An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions. A component of SCF1 is shown to be PP2Cgamma, a type 2C Ser/Thr phosphatase of previously unknown function. Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear. Here we show that human PP2Cgamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex. The phosphatase activity is required for the splicing function of PP2Cgamma, as an active site mutant does not support spliceosome assembly. The requirement for PP2Cgamma is highly specific, as the closely related phosphatase PP2Calpha cannot substitute for PP2Cgamma. Consistent with a role in splicing, PP2Cgamma localizes to the nucleus in vivo. We conclude that at least one specific dephosphorylation event catalyzed by PP2Cgamma is required for formation of the spliceosome.

Amino Acid Sequence↗

PubMed1999

Purification of bovine S100A12 from recombinant Escherichia coli.

S100A12, a member of the S100 family of EF-hand calcium-binding proteins, was purified from Escherichia coli cells expressing the corresponding cDNA. The procedure involved washing induced E. coli cells with EDTA-containing hypotonic solution, ion-exchange chromatography, and HPLC. Recombinant S100A12 was purified to homogeneity with the final yield around 6.7 mg per 20 ml of culture. The purified protein was identical to native S100A12 in the N-terminal amino acid sequence, lysylendopeptidase peptide mapping, mass spectrum, and Ca2+-dependent binding affinity to amlexanox, an antiallergy drug. However, the N-terminal methionine residue of the purified protein was not cleaved off as in the native protein. The method used in the present study permits the purification of recombinant S100A12 in large quantities and may also be applicable to preparation of other S100 family proteins.

Amino Acid Sequence↗

PubMed1999

Purification and characterization of a novel beta-agarase from an alkalophilic bacterium, Alteromonas sp. E-1.

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.

Journal Article↗

PubMed1999

Neurological complications after stem cell transplantation in childhood.

We analyzed the incidence of neurological complications in 77 patients receiving stem cell transplantation (SCT), and 12 patients (15.8%) had the following symptoms: convulsions, intracranial hemorrhage, and leukoencephalopathy. Although statistically not significant, neurological complications were seen more frequently in patients after allogeneic transplantation, and in those with acute graft-versus-host disease (GVHD) exceeding grade II. The most significant risk factor for neurological complications was identified as unrelated donor allogenic transplantation (P = 0.016). Complications were categorized into three groups, based on time of onset and symptoms: (1) convulsions during the conditioning period, (2) intracranial hemorrhage during the period of granulocyte recovery, and (3) leukoencephalopathy at around 2 months after SCT. We propose awareness of the risks of neurological complications in each period after SCT so that immediate and effective treatment of patients can be instigated.

Adolescent↗

PubMed1999

Effects of morning bright light in healthy elderly women: effects on wrist activity.

To investigate the effects of 8000 lux morning bright light in the elderly, home-based motor activity on sleep was monitored for 5 days in 10 healthy women (mean age: 59.7 years old, range: 50-69 years old). The activity level and movement index on night 4 were significantly lower in bright light conditions, compared with the controlled condition. The activity level during the day was not significantly different between the two conditions. These results indicate that 8000 lux morning bright light improves sleep quality in healthy elderly women.

Aged↗

PubMed1999

Effects of morning bright light on sleep in healthy elderly women.

Subjective sleep feeling and polysomnography were measured in 10 elderly women to investigate the effects of 8000 lux morning bright light (BL) exposure. The profile of sleep feeling in the BL condition was better than in the control condition. The proportion of awakening time in the first one-third of night sleep decreased, and the amount of awakening time in the last one-third increased in BL condition. Daytime napping reduced in BL condition. These findings suggested the effectiveness of exposure to bright light on the improvement of sleep quality and daytime vigilance of healthy elderly women.

Aged↗

PubMed1999

Analysis of rapid eye movement periodicity in narcoleptics based on maximum entropy method.

We examined REM sleep periodicity in typical narcoleptics and patients who had shown signs of a narcoleptic tetrad without HLA-DRB1*1501/DQB1*0602 or DR2 antigens, using spectral analysis based on the maximum entropy method. The REM sleep period of typical narcoleptics showed two peaks, one at 70-90 min and one at 110-130 min at night, and a single peak at around 70-90 min during the daytime. The nocturnal REM sleep period of typical narcoleptics may be composed of several different periods, one of which corresponds to that of their daytime REM sleep.

Adult↗

PubMed1999

Gender difference of slow wave sleep in middle aged and elderly subjects.

Sleep EEG of eight healthy males and eight females aged 54-72 years were recorded at their homes. The electroencephalograms were visually scored and analyzed by spectral analysis using the FFT method. There were no significant differences in sleep parameters except for a higher percentage of stage 3+4 in females. The spectral power of the delta band EEG was classified into two frequencies: 0.5-2 Hz and 2-4 Hz. The total amount of the delta band spectral power through the night was significantly larger in females. Periodic fluctuation of delta band power was observed in females along with non-rapid eye movement-rapid eye movement cycles.

Aged↗

PubMed1999

Effects of moderately bright light on subjective evaluations in healthy elderly women.

Eight healthy elderly women aged 56-72 years (mean age: 67.4 years) participated in this study. They were exposed to moderately bright light (1000 lux) for 1h in the morning every 6 days in their homes. Moderately bright light improved self evaluations for alertness, mood, motivation, happiness, refreshment, concentration and appetite after the second exposure to the light. Sleep maintenance, anxiety and integrated sleep feeling also improved after the fourth exposure to the light. The phase of rectal temperature rhythm did not change in a moderately bright light condition. These results suggest that bright light influences sleep quality and self evaluations without changing the biological rhythm phase.

Adaptation, Psychological↗

PubMed1999

Physiognomic perception, vitality affect and delusional perception in autism.

The relationship between autism and schizophrenia has been denied from the symptomatological and epidemiological standpoints. However, the mechanism whereby psychotic symptoms appear in association with autism has not been investigated at any length. Therefore, an investigation was conducted on how the unique modes of perception in autism are related to the psychotic symptoms observed. Through the therapy of one case of adolescent autism, the author points out the existence of physiognomic perception and vitality affect as characteristic modes of perception in autism. It was inferred that should autistics be placed under circumstances forcing them to withdraw from open communalism, their unique interpretation of the environmental world could give rise to psychopathological phenomena which would be considered delusional perception.

Adolescent↗

PubMed1999

A comparison of post-ictal headache between patients with occipital lobe epilepsy and temporal lobe epilepsy.

We investigated post-ictal headaches (PIH) using a questionnaire to ascertain their characteristics and compare them among different types of epilepsy. The subjects consisted of 34 patients with occipital lobe epilepsy (OLE) and 75 patients with temporal lobe epilepsy (TLE). PIH occurred in 62% of OLE and 23% of TLE (P < 0.05). The quality of pain in PIH was 'steady' in 71% of OLE and 29% of TLE (P < 0.05) as opposed to 'pounding'. Other factors, such as frequency, severity, duration, and accompanying symptoms showed no significant differences. We found very few patients with migraine-like headaches. Analyses of clinical factors, such as age at onset, duration of epilepsy, seizure frequency, family history of headache, and interictal headache did not reveal any relationship to PIH, although generalized tonic-clonic seizures are associated with PIH in TLE (P < 0.05). These results suggest that the nature of PIH may be different between OLE and TLE, and that the region of epileptic focus or spreading area of epileptic discharge may have a close relation to the induction of PIH. An association with migraine, which has been reported previously, was unclear in our study.

Adult↗

PubMed1999

Dietary thiamin level influences levels of its diphosphate form and thiamin-dependent enzymic activities of rat liver.

This study was prompted by our incomplete understanding of the mechanism responsible for the clinical benefits of pharmacological doses of thiamin in some patients with maple syrup urine disease (MSUD) and the question of whether thiamin diphosphate (TDP), a potent inhibitor of the activity of the protein kinase that phosphorylates and inactivates the isolated branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex, affects the activity state of the complex. Rats were fed a chemically-defined diet containing graded levels of thiamin (0, 0.275, 0.55, 5.5, and 55 mg thiamin/kg diet). Maximal weight gain was attained over a 3-wk period only in rats fed diets with 5.5 and 55 mg thiamin/kg. Feeding rats the thiamin-free diet for just 2 d caused loss of nearly half of the TDP from liver mitochondria. Three more days caused over 70% loss, an additional 3 wk, over 90%. Starvation for 2 d had no effect, suggesting a mechanism for conservation of TDP in this nutritional state. Mitochondrial TDP was higher in rats fed pharmacological amounts of thiamin (55 mg thiamin/kg diet) than in rats fed adequate thiamin for maximal growth. Varying dietary thiamin had marked but opposite effects on the activities of alpha-ketoglutarate dehydrogenase (alpha-KGDH) and BCKDH. Thiamin deficiency decreased alpha-KGDH activity, increased BCKDH activity, and increased the proportion of BCKDH in the active, dephosphorylated, state. Excess dietary thiamin had the opposite effects. TDP appears to be more tightly associated with alpha-KGDH than BCKDH in thiamin-deficient rats, perhaps denoting retention of alpha-KGDH activity at the expense of BCKDH activity. Thus, thiamin deficiency and excess cause large changes in mitochondrial TDP levels that have a major influence on the activities of the keto acid dehydrogenase complexes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1999

[Sepsis due to Salmonella Oranienburg--a case report].

A previously healthy 15-year-old female was admitted to our hospital complaining of nausea and vomiting. She did not complain of diarrhea. A physical examination revealed a lower right quadrant abdominal tenderness without rebound or spontaneous pain and a knocking pain of the costovertebral angle. A high fever, knocking pain of costovertebral angle, and urinary findings including Gram's stain, lead us to suspect a urinary tract infection, cefotiam was administered intravenously. Spiking fever with shaking chills continued for three days, and three sets of blood cultures were positive for Salmonella Oranienburg, but her urine culture was negative. Her history was taken again, revealing an intake of a processed squid product. The product was confirmed by the local public health center to be Salmonella Oranienburg. Finally food poisoning by Salmonella Oranienburg with sepsis was diagnosed. With cefotiam she became better and was discharged from the hospital on the 10th hospital day. During admission to the hospital she did not experience any diarrhea, and her stool culture was negative. Epidemics of Salmonella Oranienburg food poisoning are relatively rare in the literature. In Japan, one has arisen as a result of contamination of a processed squid product in March 1999. However, there have been no cases without so-called gastroenteritic symptoms (abdominal pain and diarrhea) who were previously healthy and developed sepsis caused by Salmonella Oranienburg, reported in Japan. Even in previously healthy patients, with an epidemic situation of non-typhoidal salmonellosis, salmonella sepsis must be ruled out. Among such cases, those who present with spiking fever and shaking chills should be given antibiotic therapy after taking appropriate cultures.

Adolescent↗

PubMed1999

The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases.

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.

Amino Acid Sequence↗