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Biomedical subjects

K Shishido

Publications and source records attributed to K Shishido.

At least 37 records · Page 2Linked to original sources
PubMed1998

Effect of naloxone on the bladder activity of rabbits with acute spinal injury.

BACKGROUND: Naloxone enhances bladder activity in patients with chronic spinal cord injury. However, there are few reports on naloxone for bladder morbidity in acute spinal cord injury. METHODS: We performed a prospective, controlled study of the effects of naloxone on bladder function in rabbits with and without surgical transection of the spinal cord at the 10th thoracic vertebra. Acute and chronic stages of injury were defined according to bladder function. Naloxone was given intravenously at both stages, and intrathecally at the acute stage. Bladder activity was monitored by cystometry. Blood concentrations of methionine-enkephalin were measured by radioimmunoassay. RESULTS: Spinal cord injuries were acute 1 or 2 days after surgery, and chronic after 1 or 2 weeks. Bladder capacity significantly decreased after 0.01 mg of intravenous naloxone in uninjured control rabbits, and after 0.03 mg of intravenous naloxone in rabbits with chronic-phase injuries. During the acute-injury phase, 0.3 mg of intravenous naloxone, or 0.02 mg of intrathecal naloxone, was necessary to evoke the micturition reflex. No significant changes in blood enkephalin levels were seen before or after spinal cord injury. CONCLUSION: In rabbits with acute spinal cord injury, intrathecal naloxone evoked the micturition reflex at a much lower dose than did intravenous naloxone. Intrathecal naloxone promises to become a new therapy for the acute stage of spinal cord injury for active recovery of bladder function, and could replace current therapy.

Animals↗
PubMed1998

Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA.

We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombinant plasmid pLC1-hph, which contains the L. edodes transcriptional signals and an Escherichia coli hygromycin B phosphotransferase gene. Protoplasts of L. edodes were treated by the PEG transformation mixture containing 50 units of SalI, which cleaves pLC1-hph at a single site, yielding about 15 transformants per 2.5 micrograms of DNA. The conventional PEG transformation without SalI, however, yielded only 1.5 transformants per 25 micrograms of DNA. The optimal amount of SalI for increased transformation was 50 units. In the case of transformation with SphI, which cleaves the plasmid at one site, the optimal amount of the enzyme was 2.5 units. Southern blot analysis of the SphI-derived transformants suggested that 50% of the plasmid integrations were REMI events.

Blotting, Southern↗
PubMed1997

Aggregation of yeast cells induced by the Arg-Gly-Asp motif-containing fragment of high-molecular-mass cell-adhesion protein MFBA, derived from the basidiomycetous mushroom Lentinus edodes.

A fruiting body-specific cDNA mfbAc, derived from the basidiomycete Lentinus edodes, has been shown to encode a high-molecular-mass (2157 amino acids) cell-adhesion protein MFBA containing the Arg-Gly-Asp (RGD) motif. A 425-amino-acid fragment containing the RGD motif of MFBA (designated MFBA(582-1006) peptide) produced in Escherichia coli exhibited cell-adhesion and spreading activity toward mammalian cells and cell-aggregation activity toward basidiomycetous hyphal cells via the RGD sequence. Here we investigated the biological activity of MFBA(582-1006) peptide in Saccharomyces cerevisiae. The DNA sequence encoding MFBA(582-1006) peptide, introduced into the yeast using an expression vector, resulted in a marked aggregation of the yeast cells. The aggregation was almost completely abolished by replacement of the RGD motif by an RGE motif in the peptide sequence.

Cell Adhesion Molecules↗
PubMed1997

Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

Base Sequence↗
PubMed1997

Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene.

The 565 amino-acid PRIB protein with a Zn(II)2Cys6 zinc-cluster DNA-binding motif is the expression product of the priB gene, which is most actively transcribed in an early stage of fruiting-body formation by the basidiomycete, Lentinus edodes. PRIB produced in Escherichia coli using the bacteriophage T7 expression system was purified by ion-exchange chromatographies and then subjected to random binding-site selection analysis using a pool of random 24-bp oligonucleotides with 13-bp PCR primer sites at each end. The oligonucleotides (50 bp) selected for PRIB binding were cloned into pUC19. A total of 303 cloned DNA fragments were picked randomly and sequenced. The PRIB binding sites could be grouped into 25 individual sequences, suggesting a consensus sequence of 16 bp, 5' GGGGGGGACAGGANCC 3'. Gel mobility-shift assaying of 10 randomly selected sequences all revealed a reasonable band shift. DNase I footprinting analysis of the 50-bp DNA fragment containing the sequence most similar to the consensus sequence showed that PRIB protects the entire 16-bp sequence from digestion by DNase I.

Base Sequence↗
PubMed1997

Generation of basidiomycetous hyphal cell-aggregates by addition of the Arg-Gly-Asp motif-containing fragment of high-molecular-weight cell-adhesion protein MFBA derived from the basidiomycete Lentinus edodes.

The Arg-Gly-Asp (RGD) motif-containing fragment of high-molecular-weight cell-adhesion protein MFBA derived from Lentinus edodes caused a significant aggregation of the fragmented hyphal cells of Schizophyllum commune. This fungal cell-aggregation was inhibited by a previous treatment of the cells with the Gly-Arg-Gly-Asp-Ser-Pro peptide, but not with the Gly-Arg-Gly-Glu-Ser-Pro peptide, showing that the RGD motif is essential for the cell-aggregation activity.

Basidiomycota↗
PubMed1996

The integrative transformation of Pleurotus ostreatus using bialaphos resistance as a dominant selectable marker.

A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed. When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, bialaphos-resistant colonies were obtained. This indicated that transformation was successful. Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged. A plasmid, pLC-GUS, containing the Escherichia coli beta-glucuronidase (GUS) gene under the control of the L. edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P. ostreatus with pLC-bar by co-transformation. Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transformants. These results indicated that co-transformation of P. ostreatus was successful, and that the GUS gene was expressed in P. ostreatus. This transformation system will enable us to breed commercial strains of P. ostreatus at the molecular level.

DNA, Fungal↗
PubMed1996

Evaluation of mRNAs encoding muscarinic receptor subtypes in human detrusor muscle.

PURPOSE: The present study evaluated the muscarinic receptor subtypes corresponding to m1 to m5 genes in human detrusor muscle. MATERIALS AND METHODS: The mRNAs encoding m2 and m3 subtypes were assessed by reverse transcription (RT)-polymerase chain reaction (PCR). The amounts of cDNA synthesized from m2 and m3 mRNAs were measured by using subcloned plasmid DNAs. The distribution of m2 and m3 mRNAs in detrusor was estimated by comparing the amount of m2 cDNA with that of m3 cDNA. RESULTS: The m2 mRNA:m3 mRNA ratio was 1.06:1.00 in human detrusor. In the cryostat sections of human detrusor, the presence of both m2 and m3 mRNAs was confirmed by in situ hybridization. However, the RT-PCR products derived from m1, m4 and m5 subtype mRNAs were not detected. CONCLUSION: These results suggest that human detrusor muscle coexpresses muscarinic m2 and m3 receptors and that the populations of the 2 subtypes are not significantly different.

Base Sequence↗
PubMed1995

Characterization of the promoter region of a cell-adhesion protein gene derived from the basidiomycete Lentinus edodes.

An analysis of the 2 kb nucleotide sequence including the 5'-flanking region of a cell-adhesion protein-encoding gene (mfbA) isolated from the basidiomycete Lentinus edodes revealed that the promoter region contains a TATA box, a GC box, a CAAT box, a CT-rich sequence element, a TATA box, two CT-rich sequences, and a CAAT box, in the order, from upstream to downstream. Three major and three alternative transcriptional initiation sites were located 127, 129 and 131 nucleotides and 96, 193 and 197 nucleotides downstream from the downstream TATA box, and all the three major sites are positioned just in the most downstream CT-rich sequence. Three 16 bp unique sequences similar to the binding sites of Neurospora crassa transcriptional activator protein qa-1F (Baum et al. (1987) Expression of qa-1F activator protein: Identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol. Cell. Biol. 7, 1256-1266) were present between the upstream TATA box and upstream CAAT box.

Amino Acid Sequence↗
PubMed1995

A fruiting body-specific cDNA, mfbAc, from the mushroom Lentinus edodes encodes a high-molecular-weight cell-adhesion protein containing an Arg-Gly-Asp motif.

A cDNA clone (designated mfbAc), encoding 2157 amino acids (aa), was isolated from a mature fruiting-body cDNA library of the edible mushroom Lentinus edodes. The mfbA transcript was abundant in mature fruiting bodies, detectable in immature fruiting bodies but absent in earlier developmental stages and in the vegetative mycelium. Although more abundant in the pileus than the stipe, only low levels were found in the gill tissue. The deduced MFBA protein (234.5 kDa) contained a cell-surface attachment-promoting Arg-Gly-Asp (RGD) motif. MFBA was produced in Escherichia coli using a maltose-binding protein (MBP) fusion vector, but it was cleaved into four fragments even in a protease-deficient host. A 425-aa MFBA peptide containing the RGD motif (named MFBA(582-1006) peptide) was successfully produced using the phage T7 expression system. This MFBA(582-1006) peptide exhibited a cell adhesion and spreading activity toward mammalian cells. This activity of the MFBA fragment was competitively inhibited by the Gly-Arg-Gly-Asp-Ser-Pro peptide but not by the Gly-Arg-Gly-Glu-Ser-Pro peptide, showing that the RGD motif of MFBA is essential for the cell-binding activity.

Amino Acid Sequence↗
PubMed1995

The effects and pharmacokinetics of rhG-CSF in patients with chronic renal failure.

The pharmacokinetics and effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophils and immunological function were studied in 10 patients with end-stage renal failure. A single dose and 2-week consecutive dosing of 50 micrograms/m2 of rhG-CSF were drip infused intravenously, and plasma rhG-CSF levels, peripheral blood cell counts, coagulation, and neutrophil and immunological functions were determined during treatment. The mean half-life of rhG-CSF in patients (2.47 +/- 0.64 h) was prolonged to about twice that of healthy subjects, and hemodialysis did not affect the pharmacokinetics. A marked increase in neutrophils and a slight increase in lymphocytes were observed with the single and consecutive administration of rhG-CSF, but no significant changes were noted in other leukocyte fractions and erythrocyte and platelet counts. The neutrophil alkaline phosphatase value increased significantly following rhG-CSF administration, and other neutrophil functions were also ameliorated in several patients with neutrophil dysfunction. In consecutive administration, however, mild bone pain and increased serum alkaline phosphatase were observed in about half the patients, but neither accumulation of rhG-CSF nor antibody production was detected. From these results, it is concluded that rhG-CSF is safe and effective for the treatment of neutropenia and neutrophil dysfunction in patients with renal failure.

Adult↗
PubMed1995

Presence of Active and Inactive Molecules of a Cell Wall-Associated Proteinase in Lactobacillus helveticus CP790.

Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from the gels. An elution profile of the cyanogen bromide digest of the purified 46-kDa protein obtained by reversed-phase high-performance liquid chromatography was identical to that of the 45-kDa proteinase except for one peak. An analysis of the N-terminal 21-amino-acid sequence revealed that the 46-kDa protein possesses an extra 7 amino acids at the N terminus of the 45-kDa proteinase. The 46-kDa protein was produced at constant levels during fermentation in a skim milk medium, while the 45-kDa protein was mainly observed in the middle of the exponential phase of growth and was produced in proportion to the proteinase activity. Moreover, only the 46-kDa protein was detected in the crude extract of L. helveticus CP791, a variant strain of CP790 defective in proteinase activity. These data strongly suggest that the 46-kDa protein is a precursor, inactive form of the 45-kDa proteinase.

Journal Article↗
PubMed1994

A novel cDNA, priBc, encoding a protein with a Zn(II)2Cys6 zinc cluster DNA-binding motif, derived from the basidiomycete Lentinus edodes.

A cDNA clone (designated priBc) was isolated from a primordial cDNA library of the basidiomycete, Lentinus edodes (Le). The priBc clone consisted of 2628 bp encoding 565 amino acids. As was expected, the priB transcript was abundant in primordia, while preprimordial mycelia and mature fruiting bodies contained lower levels of this Le transcript. The deduced PRIB protein (64 kDa) contained a 'Zn(II)2Cys6 zinc cluster' DNA-binding motif. PRIB was produced in Escherichia coli using the bacteriophage T7 expression system. Southwestern blot analysis revealed that PRIB binds to the DNA fragment containing the upstream region of priB.

Amino Acid Sequence↗
PubMed1994

Basidiomycetous ras cDNA functionally replaces its homolog genes in yeast.

It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomycete Lentinus edodes (named Leras cDNA) can functionally replace its homolog genes (ScRAS1 and ScRAS2) in the yeast Saccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of both RAS genes. The strain replaced by a Leras-cDNA-carrying plasmid, however, grew slower than the strains replaced by a ScRAS1- or a ScRAS2-carrying plasmid. The intracellular level of cAMP in the strain harboring the Leras-cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying the S. cerevisiae cAMP-dependent protein kinase catalytic subunit C1 gene, TPK1, but was lower than that in a strain harboring an ScRAS2-carrying plasmid. These results suggest that the Leras cDNA can complement the ras1- ras2- mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing the ScRAS2 gene.

Cyclic AMP↗
PubMed1994

Clinical study of audiograms in the initial stage and fixed stage of sudden deafness.

Audiograms in the initial stage and the fixed stage of sudden deafness were analyzed in 79 cases using the following 5 criteria; i) the onset is sudden and time of onset definite, ii) hearing loss at 1 kH is more than 35 dB, iii) the course of hearing recovery is clear, iv) unaffected ear has normal hearing, and v) the first examination and treatment is within 15 days after onset. Consequently, at least in cases receiving treatment within 6-15 days after onset, it was considered that the prognosis of hearing recovery could be established rather than the pattern of hearing loss than by the period of treatment after onset.

Adolescent↗