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K Shiba

Publications and source records attributed to K Shiba.

At least 19 recordsLinked to original sources
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

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↗
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↗
PubMed1997

Creation of libraries with long ORFs by polymerization of a microgene.

We describe a novel method for constructing pools of DNA sequences that encode large proteins with molecular diversity. Sets of primer pairs that form 8 to 10 complementary base pairs in the 3' region and have double mismatch pairs at their 3'-OH ends were designed so that primer dimers recreated short stretches of DNA (microgenes) devoid of termination codons. Cycles of denaturation and elongation reactions with a pair of primers, four dNTPs, and 3'-5' exo+ thermostable DNA polymerase gave head-to-tail polymers of the primer dimer unit (microgene) whose sizes exceeded 12 kb. No template was required in this reaction, but mismatched nucleotides at 3'-OH ends of the primers were critical for efficient polymerization. At end-joining junctions of a microgene, nucleotide insertions and deletions randomly occurred, resulting in combinatorial libraries of three reading frames from a single microgene. Further molecular diversity could be incorporated by using a mixture of primers. The resultant polymers have long ORFs whose products have a repetitious nature that could facilitate the formation of higher structures of translated products. Thus, microgene polymers may be used as a source of libraries for in vitro protein evolution experiments. Ligation of a microgene is apparently related to the nonhomologous recombination of double-strand breaks in DNA that has been shown to be catalyzed by DNA polymerases. We named this polymerization reaction the "microgene polymerization reaction."

Amino Acid Sequence↗
PubMed1997

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes.

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High-voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, kappa-chain, lambda-chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti-serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously.

Blood Protein Electrophoresis↗
PubMed1997

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion.

To clarify whether muscarinic acetylcholine receptor (mAChR) binding can be a viable muscarinic neuronal marker which provides therapeutic information different from perfusional information in global brain, we evaluated the discrepancy between the distribution of cerebral blood flow (CBF), mAChR and its five subtypes of messenger ribonucleic acid (mRNA) in the acute (n=9) and chronic (n=8) phases of a middle cerebral artery (MCA) occlusion model and in sham-operated controls (n=6). In the acute phase, regional CBF was markedly reduced in the MCA territory, whereas mAChR was not reduced and the mRNA was reduced only slightly. In the chronic phase, mAChR was reduced markedly in the infarcted lesion and the mRNA was also reduced. The mAChR was slightly reduced in the ipsilateral substantia nigra and pontine nucleus because of remote effects; however, regional CBF in the substantia nigra was slightly increased and did not change in the pontine nucleus. The discrepancy between CBF and mAChR was clarified, and the tendency toward a reduction in mRNA in the acute ischaemic region without a reduction in mAChR suggested the presence of cholinergic neurons which were viable but hypometabolic. It is concluded that mAChR imaging may be of value for the assessment of the viable cholinergic neuron density in vivo.

Animals↗
PubMed1997

The nucleus retroambigualis controls laryngeal muscle activity during vocalization in the cat.

The purpose of this study was to determine (1) whether the nucleus retroambigualis (NRA) plays an essential role in periaqueductal gray (PAG)-induced vocalization and (2) which NRA neurons are involved in the projection from the PAG to laryngeal motoneurons. Bilateral injections of the neurotoxin kainic acid into the NRA in decerebrate cats abolished PAG-induced vocalization; PAG stimulation after the injections no longer modulated vocal fold adductor or tensor activity, and only tonically, but no longer phasically, activated the abdominal muscles. In contrast, PAG-induced inspiratory excitation remained even after the injections. These results suggest that the NRA is essential for the vocal activation of the laryngeal adductor and abdominal muscles, and that an additional pathway from the PAG to respiratory motoneurons other than through the NRA is important for mediating PAG-induced inspiratory activation. Secondly, axonal projections of NRA neurons to the contralateral nucleus ambiguus (NA) were studied electrophysiologically. Five expiratory neurons, which had decrementing (n=4) or constant (n=1) firing patterns, were identified as both having axonal projections to the NA and receiving inputs from the PAG. Furthermore, following NA stimulation many constant-latency action potentials of silent cells were recorded in the vicinity of the NRA, where many silent cells were also orthodromically activated by PAG stimulation. No NRA augmenting expiratory neurons could be antidromically activated from the NA. It is suggested that the NRA and adjacent reticular formation integrate inputs from the PAG and send outputs to laryngeal motoneurons for vocalization.

Action Potentials↗
PubMed1997

Transforming growth factor beta type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer.

BACKGROUND & AIMS: Germline mutations of DNA mismatch repair genes are responsible for cancer susceptibility in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds. Transforming growth factor beta type II receptor (TGF-beta RII) has been found to be somatically altered in HNPCC. The aim of this study was to clarify further the role of TGF-beta RII alterations in HNPCC tumorigenesis, particularly in adenomas. METHODS: Fourteen adenoma specimens and 13 cancer specimens from 10 patients with HNPCC were screened for mutations in the short repeated sequences of the TGF-beta RII gene by polymerase chain reaction-single-strand conformation polymorphism. Mismatch repair genes, replication errors, and c-K-ras 2 were also analyzed in HNPCC tumors. RESULTS: Alterations of the TGF-beta RII gene at the short poly(A) repeat were found in 8 (57%) adenoma specimens and 11 (85%) cancer specimens. They were found at an earlier stage of adenomas. Two adenoma specimens showed two-hit inactivation of mismatch repair genes. Replication errors were detectable in 13 (93%) adenoma specimens. Mutations in c-K-ras 2 codon 12 were detected at a 50% frequency in adenoma specimens. CONCLUSIONS: These data indicate a strong association between TGF-beta RII gene alterations and adenoma-carcinoma progression in HNPCC.

Adult↗
PubMed1997

Influences of laryngeal afferent inputs on intralaryngeal muscle activity during vocalization in the cat.

The present study was undertaken to elucidate the possible role of the laryngeal afferent inputs in the regulation of intralaryngeal muscle activity during vocalization. We studied the influences of airflow and/or pressure applied to the larynx on intralaryngeal muscle activity during vocalization in ketamine-anesthetized cats. Vocalization was induced by airflow applied to the upper airway, which was isolated from the lower airway, during pontine call site stimulation. When the upper airway was open to the atmosphere through the nostrils and mouth, the airflow increased not only the vocal fold adductor and tensor activities but also the duration of these activities. The adductor and tensor activities were increased suddenly at a critical subglottic pressure level equivalent to the subglottic pressure threshold for vocalization. These effects were significantly reduced by sectioning of the internal branch of the superior laryngeal nerve or by lidocaine application to the laryngeal mucosa. Sustained pressure applied to the isolated upper airway, when the mouth and nostrils were occluded, did not affect adductor or tensor activities. These results indicate that the afferent inputs evoked by vocal fold stretching or vibration play an important role in the motor control of intralaryngeal and respiratory muscles during vocalization.

Afferent Pathways↗