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T Fukushige

Publications and source records attributed to T Fukushige.

At least 37 records · Page 2Linked to original sources
PubMed1999

Computed tomographic epidurography: an aid to understanding deformation of the lumbar dural sac by epidural injections.

Local anaesthetics injected into the epidural space may deform the dural sac to a variable degree, thereby contributing to variability in the extent of the block. We investigated deformation of the lumbar dural sac after injection into the lumbar epidural space. The subjects were 26 patients with low-back pain who underwent lumbar epidurography and computed tomographic (CT) epidurography, of whom seven also underwent myelography and computed tomographic myelography. The epidural space was entered via the sacral hiatus in 24 patients and through the L5/S1 interspace in two patients. Ten millilitres of local anaesthetic was then injected into the epidural space followed by 20 mL of contrast medium. Computed tomographic epidurography was undertaken approximately 30-min after the epidural injection at the mid-vertebral and mid-discal levels from the first lumbar through to the first sacral vertebrae. The dural sac usually showed an oval or hexagonal shape on the transverse views at the first and second lumbar vertebral levels, and the shape of an inverted triangle below the level of the third lumbar vertebra. A median line of translucency was also observed on the posteroanterior epidurographic view in 25 of the 26 patients. This line was though to be a manifestation of the dural deformation to the inverted triangle. Dural sac deformation usually shows a specific pattern, although there are individual variations. Dural deformability is an important consideration in any analysis of the spread of epidural block or of the changes of epidural pressure after epidural injection of local anaesthetics.

Adult↗
PubMed1999

MEC-12, an alpha-tubulin required for touch sensitivity in C. elegans.

mec-12 is one of a dozen genes required for touch receptor neuron function in Caenorhabditis elegans. Some mec-12 mutants (mechanosensory-defective) lack the large-diameter microtubules that are characteristic of these neurons (15 protofilaments, as opposed to 11). Mutants of mec-7, a alpha-tubulin encoding gene, have a similar phenotype. We have identified the nature of mec-12 by germline transformation rescue and characterization of a point mutation. Sequence analysis of the mec-12 encoded product (MEC-12) indicates that it corresponds to a novel C. elegans alpha-tubulin. MEC-12 is the only identified C. elegans alpha-tubulin that contains a lysine at position 40, a known site of post-translational acetylation. Some mec-12 mutations eliminate microtubule acetylation as assayed immunocyto-chemically; phenotypic rescue using a MEC-12 variant lacking the lysine-40 showed that acetylation is not required for MEC-12 activity. Although functionally needed only in the touch neurons, mec-12 is expressed in several other neuron types. These results support the notion that tubulin isotype diversity contributes to the formation of distinct classes of microtubules; 15-protofilament microtubule assembly requires MEC-12 alpha-tubulin and MEC-7 beta-tubulin, which are both highly expressed in the touch receptor neurons. MEC-12 is the first reported alpha-tubulin isotype that appears to be required in a single class of neuronal microtubules.

Acetylation↗
PubMed1999

[Intraoperative assessment by laser-Doppler skin blood flowmetry of the efficacy of endoscopic thoracic sympathectomy].

We have investigated whether laser-Doppler (L-D) skin blood flowmetry on the finger could be useful for an intraoperative assessment of the efficacy of endoscopic thoracic sympathectomy (ETS) under general anesthesia. Subjects were 5 young adults receiving ETS for palmar hyperhidrosis. ETS was performed with the patients in the semi-sitting position under one lung ventilation. A pair of LDF probes were placed on the palmar side of the both second fingers. Palmar hyperhidrosis disappeared after ETS in all cases, but compensatory hyperhidrosis developed in the back of the body and the thigh. After completion of ETS on one side, the L-D skin blood flow increased to 267.6 +/- 211.1% on the side of ETS, and it increased in 2 other cases and decreased on the contrary in 3 cases on the other side. After ETS on both sides the L-D skin blood flow increased to 265.0 +/- 185.9% on the side of initial ETS and to 211.4 +/- 172.8% on the side of subsequent ETS. The initial EST induced reflex vasoconstriction on the finger of both sides and also on the toe. Spontaneous fluctuation and reflex vasoconstriction of the skin blood flow were still observed, although the periodicity of spontaneous fluctuation between the right and the left finger was lost in some of the cases. An increase in L-D skin blood flow on the side of ongoing ETS is useful for intraoperative assessment of ETS.

Adult↗
PubMed1998

Reprogramming of early embryonic blastomeres into endodermal progenitors by a Caenorhabditis elegans GATA factor.

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development.

Animals↗
PubMed1998

The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine.

The Caenorhabditis elegans elt-2 gene encodes a single-finger GATA factor, previously cloned by virtue of its binding to a tandem pair of GATA sites that control the gut-specific ges-1 esterase gene. In the present paper, we show that elt-2 expression is completely gut specific, beginning when the embryonic gut has only two cells (one cell cycle prior to ges-1 expression) and continuing in every cell of the gut throughout the life of the worm. When elt-2 is expressed ectopically using a transgenic heat-shock construct, the endogenous ges-1 gene is now expressed in most if not all cells of the embryo; several other gut markers (including a transgenic elt-2-promoter::lacZ reporter construct designed to test for elt-2 autoregulation) are also expressed ectopically in the same experiment. These effects are specific in that two other C. elegans GATA factors (elt-1 and elt-3) do not cause ectopic gut gene expression. An imprecise transposon excision was identified that removes the entire elt-2 coding region. Homozygous elt-2 null mutants die at the L1 larval stage with an apparent malformation or degeneration of gut cells. Although the loss of elt-2 function has major consequences for later gut morphogenesis and function, mutant embryos still express ges-1. We suggest that elt-2 is part of a redundant network of genes that controls embryonic gut development; other factors may be able to compensate for elt-2 loss in the earlier stages of gut development but not in later stages. We discuss whether elements of this regulatory network may be conserved in all metazoa.

Animals↗
PubMed1998

pha-4 is Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma homolog that functions in organogenesis of the C. elegans pharynx.

The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila fork head and mammalian HNF-3alpha,beta,gamma genes, and mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. In the present paper, we show that Ce-fkh-1 and pha-4 are the same gene. We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells, of all five cell types. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independently of cell type. We show that PHA-4 binds directly to a 'pan-pharyngeal enhancer element' previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. We also show that ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. We propose that it is the combination of pha-4 and regulatory molecules such as ceh-22 that produces the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an 'organ identity factor', completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. Finally, we note that the distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the fork head protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. Moreover, we show that pha-4 expression in the C. elegans gut is regulated by elt-2, a C. elegans gut-specific GATA-factor and possible homolog of the Drosophila gene serpent, which influences fork head expression in the fly gut. Overall, our results provide evidence for a highly conserved pathway regulating formation of the digestive tract in all (triploblastic) metazoa.

Animals↗
PubMed1996

Modulation of gene expression in the embryonic digestive tract of C. elegans.

The Caenorhabditis elegans digestive tract is composed of four distinct modules derived from separate cell lineages: anterior pharynx from the ABa lineage, posterior pharynx from the MS lineage, gut from the E lineage, and rectum from the ABp lineage. The C. elegans gut esterase gene (ges-1) is normally expressed in the embryonic gut or E lineage. However, expression ges-1 can be switched into cells of the embryonic pharynx and tail by virtue of deleting a tandem pair of WGATAR sites in the ges-1 promoter. Here, we use both laser ablation experiments and genetic analysis to show that cells expressing the WGATAR-deleted ges-1 transgene belong to all three nongut lineages of the digestive tract: ABa, MS, and ABp. We also show that the molecular size and spatial distribution of ges-1 mRNA transcripts produced by either the WGATAR-deleted ges-1 transgene or the undeleted ges-1 control transgene appear correctly regulated, suggesting that the spatial switch in ges-1 expression occurs at the level of transcription initiation. We further show that both the WGATAR-deleted and the undeleted ges-1 transgenes respond appropriately to mutations in a series of maternal effect genes (skn-1, mex-1, pie-1, and pop-1) that alter early blastomere fate. Moreover, the pharynx/tail expression of the WGATAR-deleted ges-1 transgene is abolished by mutations in the zygotic gene pha-4. Finally, we use imprecise transposon excision to produce two independent C. elegans strains with 1- to 2-kb deletions that remove the tandem WGATAR sites from the promoter of the endogenous chromosomal ges-1 gene: in both of these strains, ges-1 is not expressed in the embryonic gut but is expressed in cells of the embryonic pharynx; pharynx expression is weak but incontrovertible. Overall, our results validate previous transgenic analysis of ges-1 control and show further that ges-1 appears to be regulated in a system-specific, rather than a lineage-specific, manner. The multiple facets of ges-1 expression provide an opportunity to investigate how a multicomponent organ system such as the digestive tract is established from diverse cell lineages.

Animals↗
PubMed1996

A high performance system for molecular dynamics simulation of biomolecules using a special-purpose computer.

GRAPE (GRavity PipE) processors are special purpose computers for simulation of classical particles. The performance of MD-GRAPE, one of the GRAPEs developed for molecular dynamics, was investigated. The effective speed of MD-GRAPE was equivalent to approximately 6 Gflops. The precision of MD-GRAPE was good judging from the acceptable fluctuation of the total energy. Then a software named PEACH (Program for Energetic Analysis of bioCHemical molecules) was developed for molecular dynamics of biomolecules in combination with MD-GRAPE. Molecular dynamics simulation was performed for several protein-solvent systems with different sizes. Simulation of the largest system investigated (27,000 atoms) took only 5 sec/step. Thus, the PEACH-GRAPE system is expected to be useful in accurate and reliable simulation of large biomolecules.

Computational Biology↗
PubMed1995

Molecular cloning and expression of serum calcium-decreasing factor (caldecrin).

We previously reported on the purification of a serum calcium-decreasing factor, referred to as caldecrin, from porcine pancreas, that is thought to be a serine protease (Tomomura, A., Fukushige, T., Noda, T., Noikura, T., and Saheki, T. (1992) FEBS Lett. 301, 277-281). In the present study, we purified caldecrin from rat pancreas and determined its primary structure by cDNA cloning. The predicted caldecrin protein is presumed to be synthesized as a preproenzyme of 268 amino acids with a signal peptide of 16 amino acids and an activation peptide of 13 amino acids, and is, with the exception of a central region, almost identical to the reported rat pancreatic elastase IV sequence. The caldecrin gene is selectively expressed in the pancreas, as judged by Northern blot analysis. After expression in BMT-10 cells, immunoreactive caldecrin was found in the culture supernatant, and it inhibited the parathyroid hormone-stimulated 45Ca release from cultured fetal long bones. Catalytic site mutants were synthesized in a baculovirus system, and recombinant mutants also decreased the serum calcium level of mice. These data implicate caldecrin, a protease closely related to elastase IV, in the regulation of blood calcium levels.

Amino Acid Sequence↗
PubMed1995

Selective expression of the tba-1 alpha tubulin gene in a set of mechanosensory and motor neurons during the development of Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, a monoclonal antibody 3A5 raised against Drosophila alpha tubulins selectively stains the nervous system immuno-cytochemically. Direct screening of a C. elegans cDNA expression library with 3A5 has allowed cloning of the tba-1 (tubulin alpha-1) gene from C. elegans. The corresponding genomic DNA encodes a protein of 449 amino acid residues that has a high homology with the vertebrate alpha tubulins but a lower homology with yeast alpha tubulins. Interestingly, the carboxyl-terminus sequence EEEGEEY (Glu-Glu-Glu-Gly-Glu-Glu-Tyr) of the nematode tba-1 encoded isotype is identical to these residues in human, mouse, rat, pig and chicken alpha-1 tubulin isotypes that are expressed in the brain. Temporal and spatial expression studies of the tba-1 gene using Northern blot analysis and tba-1::lacZ fusion gene expression analysis during embryonic and the postembryonic development of C. elegans reveal that the tba-1 tubulin is preferentially expressed in the nematode nervous system, especially in a set of mechanosensory neurons and a set of ventral cord motor neurons (DA, DB, VA, and VB) during embryonic and postembryonic development. Our results indicate an inter-species conservation of the alpha tubulin carboxyl-terminal domain in functionally related brain specific isotypes from metazoans as divergent as mammals and nematodes. These results also suggest specificity of the individual alpha tubulin isotypes during neural development.

Amino Acid Sequence↗
PubMed1995

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia.

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6(-2) (a transition of A to G at the second nucleotide position within the 3' splice cleavage site of intron 6) and IVS-13(+5) (a transition of G to A at the fifth nucleotide position within the 5' splice cleavage site of intron 13), respectively. The IVS-6(-2) mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6(-2) mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Effect of the dpy-20 and rol-6 cotransformation markers on alpha-tubulin gene expression in C. elegans transformants.

An alpha-1 tubulin::lacZ fusion gene was introduced into the germline of Caenorhabditis elegans, using either rol-6 or dpy-20 genomic DNA as a cotransformation marker. Distinct patterns in cellular specificity of the alpha-1 tubulin::lacZ fusion gene expression were observed, depending on the cotransformation marker used. For the rol-6 marker, the tubulin fusion gene was expressed in several neurons in the head and tail ganglia and a set of 38-39 ventral cord motor neurons along the body length of the animal during larval and adult development. In contrast, for the dpy-20 marker system, not only were fewer neurons stained in the head and tail region, but also the staining of ventral cord motor neurons was extremely reduced both in number and intensity. The dpy-20 marked-mediated suppression of the alpha-1 tubulin gene expression was observed both in the cis and trans configurations. Similar down-regulation in the ventral cord motor neurons was observed when the alpha-2 tubulin::lacZ fusion gene construct was tested in these experiments using the dpy-20 marker. In controls, where the tubulin fusion gene was directly injected to obtain transformants without any marker DNA, the cellular staining pattern was close to the fusion gene expression observed with the rol-6 marker DNA. These results underline the importance of the choice of transformation marker system in generation of the transgenic animals, and reveal a down-regulation of the alpha-tubulin fusion gene expression in the ventral cord motor neurons in transgenic animals when the dpy-20 gene was used as a cotransformation marker.

Animals↗
PubMed1994

A special-purpose computer for molecular dynamics: GRAPE-2A.

Molecular dynamics simulations have been extensively used in research of proteins. Since these simulations are quite computer intensive, their acceleration is of main interest of the research. In molecular dynamics simulations, almost all computing time is consumed in calculating the forces between particles, e.g., Coulomb and van der Waals forces. We have designed and built GRAPE-2A (GRAvity PipE 2A), a special-purpose computer for use in simulations of classical many-body systems. GRAPE-2A calculates forces exerted on a particle from the other particles. GRAPE-2A can calculate force of an arbitrary functional form of a central force. The host computer, which is connected to GRAPE-2A through the VME bus, performs other calculations such as time integration. The peak speed of GRAPE-2A is 180 Mflops. We can also stimulate systems with periodic boundary conditions by the Ewald method, using GRAPE-2A and another special-purpose computer, WINE (Wave space INtegrator for the Ewald method).

Computer Simulation↗
PubMed1993

Molecular cloning and developmental expression of the alpha-2 tubulin gene of Caenorhabditis elegans.

Alpha tubulin isotypes are encoded by at least four genes designated alpha-1 to alpha-4 in the nematode Caenorhabditis elegans. We describe here, molecular cloning of the alpha-2 tubulin gene, located on chromosome I, that encodes a protein of 449 amino acids that has high homology to human, mouse and Drosophila alpha tubulins, but relatively lower homology to the yeast alpha tubulins. The alpha-2 tubulin gene is trans-spliced to the SL1 leader sequence. Northern analysis shows that the gene is increasingly transcribed during the early (L1-L3) larval stages but has a lower level of transcription in L4 L4 larvae, adults, and embryos. Using an alpha-2-lacZ fusion gene expression in transgenic animals, we show that the gene is expressed in a tissue-specific manner in the intestine, pharyngeal muscle cells, and a subset of neurons which include a class of DB and VB motor neurons in the ventral nerve cord, posterior touch receptor neurons, PLML, PLMR, in the lumbar ganglia; PVT in the pre-anal ganglion, and ALA in the dorsal ganglion in the head. Our results support the notion that tubulin structure may contribute to the functional specialization of microtubules.

Amino Acid Sequence↗
PubMed1993

Caldecrin proform requires trypsin activation for the acquisition of serum calcium-decreasing activity.

Proform serum calcium-decreasing factor (procaldecrin) was purified from porcine pancreas acetone powder. Procaldecrin showed chymotrypsin activity after trypsin treatment in a time- and dose-dependent manner. Procaldecrin did not possess serum calcium-decreasing activity but acquired serum calcium-decreasing activity as well as protease activity after trypsin treatment. However, PMSF treatment after activation of procaldecrin by trypsin did not affect the serum calcium-decreasing activity, even though protease activity was nullified by treatment with PMSF. These findings suggest that the serum calcium-decreasing activity acquired by procaldecrin requires conformational change caused by trypsin treatment.

Amino Acid Sequence↗