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K Shiba

Publications and source records attributed to K Shiba.

At least 55 records · Page 3Linked to original sources
PubMed1998

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1998

Strong selective pressure to use G:U to mark an RNA acceptor stem for alanine.

The identity of alanine tRNAs is dependent on a G:U base pair at the 3:70 position of the acceptor helix. This system of molecular recognition is widely distributed from bacteria to human-cell cytoplasm. In contrast, some mitochondrial alanine acceptor helices are markedly different and contain nucleotides known to block aminoacylation by a nonmitochondrial enzyme. Thus, acceptor helix recognition may differ in these systems and may not depend on G:U. Here we report an example of a Caenorhabditis elegans mitochondrial system where the G:U pair is preserved but where proximal nucleotides known to block charging by a nonmitochondrial enzyme are also present. We show that, as expected, the mitochondrial substrate is not charged by the bacterial enzyme. In contrast, the cloned mitochondrial enzyme charged both mitochondrial and bacterial microhelices. Strikingly, charging of each required the G:U pair. Thus, G:U recognition persists even with an acceptor helix context that inactivates nonmitochondrial systems. The results suggest strong selective pressure to use G:U in a variety of contexts to mark an acceptor stem for alanine. Separate experiments also demonstrate that, at least for the mitochondrial enzyme, helix instability or irregularity is not important for recognition of G:U.

Acylation↗
PubMed1998

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro.

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Acylation↗
PubMed1998

Biochemical and phylogenetic analyses of methionyl-tRNA synthetase isolated from a pathogenic microorganism, Mycobacterium tuberculosis.

Mycobacterium tuberculosis methionyl-tRNA synthetase (MetRS) has been cloned and characterized. The protein contains class I signature sequences but lacks the Zn2+ binding motif and the C-terminal dimerization appendix that are found in MetRSs from several organisms including E. coli MetRS. Consistent with these features, the enzyme behaved as a monomer in a gel filtration chromatography and did not contain the bound Zn2+. Nonetheless, it was active to the tRNAMet of E. coli as determined by in vivo genetic complementation and in vitro reaction. Phylogenetic analysis separated the M. tuberculosis and E. coli MetRSs into prokaryote and eukaryote-archaea group, respectively. This result is consistent with the taxonomic locations of the organism but is an interesting contrast to the case of its paralogous protein, isoleucyl-tRNA synthetase, and suggests that the two enzymes evolved in separate idiosyncratic pathways.

Amino Acid Sequence↗
PubMed1998

Upper airway motor outputs during vomiting versus swallowing in the decerebrate cat.

Swallowing and vomiting are antagonistic motor acts; nevertheless, vomiting can be immediately followed by swallowing. The purpose of this study was to clarify the interrelationship between these two behaviors, particularly in regard to comparing the upper airway motor patterns at the end of the expulsion phase with those during subsequent swallowing. Experiments were conducted using both paralyzed and non-paralyzed decerebrate cats, in which recordings were obtained either from upper airway muscles, the diaphragm and abdominal muscles or from the nerves that innervate those muscles. The activity patterns of most nerves recorded in paralyzed animals were consistent with the behavior recorded in non-paralyzed animals from the muscles innervated by those nerves, with the exception of the cricothyroid and stylopharyngeus muscles. Vomiting can be divided into a series of retches followed by expulsion, which itself can be further subdivided into three phases. The final stage of expulsion, characterized by burst-like exaggerated activity of the laryngeal elevator thyrohyoid and the pharyngeal constrictors, proved to be different from pharyngeal swallowing, as judged from differences in the spatio-temporal patterns of the upper airway motor outputs. However, post-vomiting swallowing activity was still observed even after total deafferentation of the laryngeal and pharyngeal areas in paralyzed animals. It is therefore likely that the central processes for vomiting and swallowing closely relate in generating these two behaviors.

Afferent Pathways↗
PubMed1998

Retrograde transport of nerve growth factor from olfactory bulb to olfactory epithelium.

NGF is produced in the olfactory bulb and transported from the bulb to cholinergic neurons in the horizontal limb of the diagonal band. Although the expression of NGF receptors has been reported in olfactory epithelial cells, no correlation between NGF and olfactory epithelial cells has been found. This study aimed to define whether or not retrograde transport of NGF occurs in the olfactory neural system using the method of radioluminography. 125I-labeled NGF injected into the olfactory bulb was taken up and transported to the olfactory epithelium 18 h after injection. This finding suggests that bulbar NGF may act as a neurotrophic factor in olfactory epithelial cells.

Animals↗
PubMed1998

Recovery of retching after lesions involving the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) in the caudal brainstem receives various inputs that trigger vomiting. Chemical (kainic acid) NTS lesions in decerebrate, paralyzed cats temporarily suppressed fictive vomiting induced by different emetics. Subsequently, retching but not expulsion, resumed in 2-3.5 h. Since the NTS does not appear essential for retching, antiemetic drugs targeted at the NTS may not completely suppress vomiting.

Animals↗
PubMed1998

Upper airway motor outputs during sneezing and coughing in decerebrate cats.

The purposes of the present study were to determine which upper airway movements cause a difference in the expiratory airflow pathway between sneezing and coughing, and to develop a new animal model for studying the neural mechanism of sneezing in paralyzed animals, i.e. fictive sneezing. We compared the upper airway motor patterns of sneezing and coughing, induced by electrical stimulation of the anterior ethmoidal nerve (AEN) and superior laryngeal nerve, respectively, in non-paralyzed decerebrate cats. Respiratory and laryngeal motor patterns that consisted of an inspiration phase, compression phase, and expulsion phase were observed for both sneezing and coughing. The main difference was observed in the activity of the elevator of the back of the tongue, styloglossus (SG) muscle, which was explosively activated during the expulsion phase of sneezing, whereas it was virtually silent during coughing. The nasopharyngeal closers were weakly to moderately activated during sneezing. Their activities during coughing were weaker than during sneezing. Furthermore, the AEN-induced activities of the phrenic and abdominal nerves and the lateral branch of the hypoglossal nerve (lat-XII), which innervates the SG muscle, in paralyzed cats were consistent with the activities of the diaphragm, abdominal, and SG muscles during actual sneezing in non-paralyzed cats. Thus, we conclude that tongue movement is the main difference in the motor outputs between sneezing and coughing, which probably causes greater nasal airflow in sneezing, and that it is necessary to record the activity of the lat-XII to identify fictive sneezing in paralyzed cats.

Abdominal Muscles↗
PubMed1998

[A clinical investigation of bacteremia for the past ten years at the Second Department of Internal Medicine, Jikei University Hospital].

We clinically investigated a total of 288 cases of bacteremia for the past ten years, from January 1986 to December 1995, at the Second Department of Internal Medicine in the jikei University Hospital. All of the subjects who had a positive reaction to blood culture or catheter tip culture were investigated for their basic disease, complications, and detected bacteria. Malignant tumors, chronic renal failure, diabetes mellitus, and hematologic disease were frequent by noted. The cases due to primary infection were mainly respiratory organ infection or urinary tract infection, which were 47.8% of the total. In 31.3% of the total, catheter tip cultures were positive. Except for catheter related infection, Gram-positive coccus were detected in 40.3%, which was most frequent. Methicillin resistant Staphylococcus aureus (MRSA) were 8.1% and Staphylococcus epidermidis were 11.2%. In catheter related infection, Gram-positive coccus were detected in 59.9%, which was most frequent amongst them, MRSA was 17.2%, S. epidermidis was 16.2%. The mortality of bacteremia was 12.5%, mainly from hematologic diseases, immunodeficiency due to long term steroid administration etc. Accordingly, the more the advance of chemotherapy, the better the prognosis of septicemia is. Appearance of catheter related infection was unexpected frequent. Increase of immunocompromised host is thought to be one of the main factors in the outbreak of bacteremia.

Adolescent↗
PubMed1998

The rapid method in microbiology using chemical method and immunochemical method.

In recent years, biochemical and immunochemical procedures used for rapid diagnosis of microbiology have advanced. In particular, methods using immunochemical procedure are commonly used. Latex agglutination test, enzyme immunoassay and immunochromatographic methods having more simple and high sensitivity have also been used. These methods are routinely used to detect Helicobacter pylori, Group A Streptococcus and others. Furthermore, in routine examinations, endotoxin and (1two head right arrow3)-beta-D-glucan have been measured as markers that detect gram-negative bacteria and deep fungus. Currently, there are many infections including new and previous infections. These techniques should be applied for detecting a larger number of microbiology.

Cryptococcus↗
PubMed1998

Use of the highly sensitive PCR method to detect the Herpes simplex virus type 1 genome and its expression in samples from Behçet disease patients.

To study the involvement of the Herpes simplex virus type 1 (HSV-1) in patients with Behçet's disease (BD), PCR was used to detect HSV-1 DNA and RNA (LAT, TK, gB) in peripheral blood leukocytes and oral smears from patients with BD. Out of 32 patients with BD and 30 healthy volunteers, HSV-1 specific DNA was not detected in any case. Using a highly sensitive competitive RT-PCR method, HSV-1 specific mRNA was not detected either in peripheral blood leukocytes from any of the patients or volunteers. In contrast, serum levels of IgG anti HSV-1 antibody were higher in patients with BD; however, there were 70% sero-positives among the control subjects. Even though it has been reported that the highly frequent presence of the HSV-1 genome in peripheral blood is a characteristic feature of BD, our results show that the HSV-1 genome and its expression are not present in peripheral blood in patients with BD. We could not find any evidence that the lesions associated with BD were caused by the direct lytic effects of HSV-1.

Adult↗
PubMed1998

A family with high serum leucine aminopeptidase activity derived from a novel variant CD13.

We investigated a family in which some individuals showed extremely high serum leucine aminopeptidase (LAP) (EC 3.4.11.2) activity, mainly derived from a variant CD13. The isoelectric points of the variant and normal CD13 were 3.3 and 4.1, respectively, and both points converged at 4.4 after treatment with neuraminidase, indicating that more sialic acids are bound to the variant CD13 than normal. The molecular masses of both CD13s were 144 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment with neuraminidase, N-glycosidase, and O-glycosidase, apparent molecular masses of the variant and normal CD13 were 106 kDa and 100 kDa, respectively, suggesting that the variant CD13 contains a longer peptide than normal. This is the first case of familial high serum LAP activity in which the origin could be demonstrated by anti-CD13 monoclonal antibodies to be a variant CD13 inherited in an autosomal dominant mode. The isoelectric point of the LAP activity after neuraminidase treatment was different from that previously reported.

Adult↗
PubMed1998

Analysis of urinary albumin, transferrin, N-acetyl-beta-D-glucosaminidase and beta2-microglobulin in patients with impaired glucose tolerance.

We investigated the changes in urinary albumin and urinary transferrin as glomerular proteins, and in urinary N-acetyl-beta-D-glucosaminidase and urinary beta2-microglobulin as tubular proteins, in patients with impaired glucose tolerance. We attempted to compare the proteins of normal subjects to those of diabetics with pre-nephropathy. Transferrin and N-acetyl-beta-D-glucosaminidase levels were significantly increased in patients with impaired glucose tolerance, while albumin and beta2-microglobulin levels were only slightly increased. In addition, there was no significant difference in transferrin levels between patients with impaired glucose tolerance and type 2 diabetics with pre-nephropathy. In our observation, although albumin levels were only slightly increased in patients with impaired glucose tolerance, a sharp increase in transferrin levels was reflected in patients with glomerular disorders. In addition, since N-acetyl-beta-D-glucosaminidase levels varied markedly, tubular disorders were suspected. It should be stressed that increased parameters for both glomerular and tubular disorders in group C--patients who showed abnormal levels in three proteins--had already been observed in some patients with impaired glucose tolerance. Therefore, the evaluation of the mutual relationships between various urinary protein components in patients with impaired glucose tolerance will become a more important assessment tool than that of single urinary protein components.

Acetylglucosaminidase↗
PubMed1997

Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene.

Familial adenomatous polyposis coli (FAP) is a disease characterized by the development of multiple colorectal adenomas, and affected individuals carry germline mutations in the APC gene. With the use of a conditional gene targeting system, a mouse model of FAP was created that circumvents the embryonic lethality of Apc deficiency and directs Apc inactivation specifically to the colorectal epithelium. loxP sites were inserted into the introns around Apc exon 14, and the resultant mutant allele (Apc580S) was introduced into the mouse germline. Mice homozygous for Apc580S were normal; however, upon infection of the colorectal region with an adenovirus encoding the Cre recombinase, the mice developed adenomas within 4 weeks. The adenomas showed deletion of Apc exon 14, indicating that the loss of Apc function was caused by Cre-loxP-mediated recombination.

Adenomatous Polyposis Coli↗
PubMed1997

Role of nucleus retroambigualis in respiratory reflexes evoked by superior laryngeal and vestibular nerve afferents and in emesis.

An ascending projection from the medullary nucleus retroambigualis (NRA) has recently been described as important for the control of the upper airway during vocalization. We evaluated the importance of this projection in other behaviors by making localized injections of the neurotoxin kainic acid in the NRA in decerebrate cats, most of which were paralyzed and artificially ventilated. In contrast to its importance for vocalization, the NRA is not essential for activation of upper airway musculature during respiration, swallowing, vomiting, or reflexes elicited by superior laryngeal or vestibular nerve afferents. However, kainic acid injections in the NRA and adjacent reticular formation prolonged the inhibitory phrenic motoneuronal response to superior laryngeal nerve stimulation and abolished or reduced abdominal motoneuronal responses during respiration, vomiting, and superior laryngeal nerve stimulation. Thus, of the behaviors we investigated, the importance of the ascending projection from the NRA appears to be limited to vocalization, while descending projections from the NRA region are important in a number of behaviors.

Animals↗
PubMed1997

Human lysyl-tRNA synthetase accepts nucleotide 73 variants and rescues Escherichia coli double-defective mutant.

The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known. The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.

Amino Acid Sequence↗
PubMed1997

A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2.

Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.

Amino Acid Sequence↗