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Biomedical subjects

H Taguchi

Publications and source records attributed to H Taguchi.

At least 37 records · Page 2Linked to original sources

Structural organization of the pigment cell-specific gene located at the brown locus in mouse. Its promoter activity and alternatively spliced transcript.

The pigment cell-specific gene, located at the brown (b) locus in mouse, has been cloned and characterized. Its gene product is required for the formation of black melanin rather than brown, although its exact function remains to be elucidated. We thus tentatively named it b-locus protein in this report. The b-locus protein gene is about 18 kilobase pairs long and organized into 8 exons and 7 introns. Functional analysis of its promoter region suggests that the nucleotide residues -38/154 is sufficient to direct the pigment cell-specific transcription in melanoma whole cell extracts. On the other hand, we were unable to detect its transcripts in HeLa whole cell extracts. Sequence comparison with the promoter region of the tyrosinase gene, another pigment cell-specific gene, reveals that two elements of the b-locus protein gene (-33/-24 and 18/28) are also conserved in the tyrosinase gene at equivalent positions, suggesting that these two elements may be involved in their pigment cell-specific transcription. Furthermore, we have cloned a cDNA, pMT3, coding for an isoform of b-locus protein from a cDNA library of mouse B16 melanoma cells. Sequence analysis of pMT3 reveals a deletion of 103 base pairs, which corresponds to the 5'-end of the exon 8 of the b-locus protein gene, indicating that pMT3 represents a mRNA species generated by alternative splicing. Since this deletion changes the reading frame and eliminates the transmembrane domain of b-locus protein, the pMT3-type mRNA may code for a soluble isoform. Such an isoform, consisting of 553 amino acids, differs only in its carboxyl terminus and is larger than b-locus protein by 16 amino acids. Using transient expression assays, we confirmed that such an isoform is able to react with anti-b-locus protein monoclonal antibody, TMH-1, suggesting that a b-locus protein isoform may have some function in pigmentation.

Amino Acid Sequence

D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum.

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.

Alcohol Oxidoreductases

Immunoglobulin prophylaxis against milkborne transmission of human T cell leukemia virus type I in rabbits.

Prophylactic effect of human T cell leukemia virus type I (HTLV-I) immune globulin (HTLVIG) against milkborne transmission of HTLV-I was investigated in a rabbit model. Four litters (A-D: 7, 5, 7, and 7 offspring, respectively) born to an HTLV-I-infected rabbit were used. Litters A and D were allowed to grow normally as controls, while litters B and C were given weekly intraperitoneal inoculation of HTLVIG four times until weaning at 4.5 weeks of age. Only 1 (8.3%) of the 12 HTLVIG-inoculated rabbits, compared with 6 (42.9%) of the 14 control rabbits, seroconverted for HTLV-I. Gene amplification detected the presence of HTLV-I proviral sequences in all of the seroconverted but in none of the seronegative rabbits. These results suggest that passive immunization is effective in preventing dam-to-offspring transmission of HTLV-I.

Animals

Serologic and ultrasonographic parameters of praziquantel treatment of hepatic fibrosis in Schistosoma japonicum infection.

We describe the parameters useful in evaluating the development of hepatic fibrosis in Schistosoma japonicum infection, as well as its improvement after treatment with praziquantel (PZQ). Various serologic parameters and ultrasonographic images were examined, and their changes were monitored using rabbits infected with 200 or 300 cercariae of S. japonicum. Infected rabbits were administered one oral treatment of PZQ at a dosage of 100 mg/kg at 6, 12, or 24 weeks after infection. Histopathologic examinations revealed that PZQ had a strong and rapid effect, even on damage that developed long after the infection. The improvement of moderate hepatic fibrosis that developed over 24 weeks after infection was also detected by histopathologic examinations. The serum level of total bile acid was the most sensitive parameter in evaluating the severity of hepatic fibrosis and its improvement after treatment with PZQ. The level of serum procollagen-III-peptide was also useful in evaluating the development of hepatic fibrosis, but not in its improvement. Ultrasonography revealed specific echogenic bands and nodules according to the progress of granuloma formation and fibrosis, and the reversal of these changes could also be observed after treatment with PZQ.

Alanine Transaminase

Image analysis by electron microscopy of two-dimensional crystals developed on a mercury surface of chaperonin from Thermus thermophilus.

Two-dimensional crystals of functional chaperonin molecules, which are protein complexes of cpn60 and cpn10, isolated from Thermus thermophilus were prepared on a mercury surface under oxygen atmosphere and were observed by electron microscope after transferring them to carbon coated specimen grids. The crystals showed the hexagonal lattice with unit cell dimensions of a = b = 12.4 nm and gamma = 120 degrees. The averaged image at a 3 nm resolution of the chaperonin has a doughnut-like shape which has seven peripheral masses and a central cavity. Preincubation of the chaperonin with MgATP changed the mobility in non-denaturating PAGE but did not cause distinguishable change of shape. The location of cpn10 in the chaperonin molecule is discussed.

Bacterial Proteins

[A comparison of measurements of serum free thyroxine concentration by equilibrium dialysis and ultrafiltration using undiluted serum].

We have examined the clinical usefulness for free thyroxine (FT4) measurement by equilibrium dialysis of undiluted serum and by radio-immunoassay of T4 concentration in the dialysate (ED/RIA). The results were compared with those by ultrafiltration of undiluted serum (UF/RIA), which is believed to offer the best possible approach towards estimation of in vitro serum level of FT4. The precision with both methods was excellent. There was a high degree of correlation of FT4 concentration by both methods (r = 0.831, p less than 0.001, n = 99). FT4 concentration by ED/RIA and UF/RIA methods agreed well for hyperthyroidism, hypothyroidism, normal pregnancy, and for low T4-binding globulin concentration. In nonthyroidal illness (NTI), FT4 levels were all within or above normal for UF/ED method, while some FT4 values in low T4 syndrome were below normal for ED/RIA method. The effect of progressive serum dilution on FT4 concentration was also examined for both methods. There was no fall in FT4 concentration in sera from normal, hyperthyroid patient and in serum with low T4 syndrome for both methods, but in a serum from low T3 syndrome, there was a fall in FT4 concentration by dilution with UF/RIA method, indicating that serum dilution with ED/RIA method might underestimate FT4 concentration in some NTI sera. From these results it is concluded that the present ED/RIA method is adaptable to clinical laboratory use as a reference standard method for FT4 measurement.

Adult

[Analgesic effects of epidural morphine, fentanyl and lidocaine].

The analgesic effects of epidurally administered morphine 5 mg (group M, n = 15), fentanyl 100 micrograms (group F, n = 15), 2% lidocaine 60 mg (group L, n = 15) and normal saline (group S, n = 10) were investigated in 55 patients scheduled for abdominal surgery. Each drug was prepared in 3 ml solution and was injected though an epidural catheter introduced 3 cm cephalad into the epidural space at T10-11. Analgesic effects were assessed by changes in the dull pain sensation induced by electrical stimulation at 3 Hz through a pair of stainless needles which were placed subcutaneously at T7 and T10 dermatomes. In group M, analgesic effects at T10 were demonstrated in 12 of 15 subjects and the onset of analgesia was more rapid at T10 than at T7. The mean onset time of analgesia was 7.8 +/- 3.6 (mean +/- SD) min. There were 5 subjects in group F and 6 in group L who showed more rapid onset of analgesic effects at T10 than at T7, respectively. There were 2 subjects in group F and 5 in group L, with more rapid onset of analgesia at T7 than at T10. There were several subjects in group F and L with simultaneous onset of analgesia at T7 and T10. In group L, the mean distribution of analgesic area, confirmed with pinprick, was 5.2 +/- 1.9 (mean +/- SD) dermatomal segments. Hypercapnea, associated with somnolence, was frequently seen in group F. None of the subjects in group M, L or S showed such incidents. These results suggest that the main site of action of epidural morphine is located in the spinal cord while that of epidural fentanyl in the brain.

Adult

Isochromosome 11q in acute monoblastic leukemia (M5a).

An isochromosome 11q in a patient with M5a type acute monoblastic leukemia is reported. The leukemic cells had a few azurophilic and alpha-naphthyl butyrate esterase-positive granules in the cytoplasm. Electron microscopy showed indented nuclei, abundant perinuclear fibrous bundles, and small lysosomes which were characteristic of monocytoid cells. A strong inhibitory activity against urokinase was detected in the cell lysates as compared with other leukemic cells. Cytogenetic analysis of the leukemic cells showed the karyotype 47, XY, +i(11q), which has not been observed hitherto in acute monocytic leukemia.

Chromosome Aberrations

Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein.

Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.

Antigens, Bacterial

In vitro IgG rheumatoid factor production by CD5-negative murine B cells in response to immune complexes of lipopolysaccharide.

We studied the in vitro production of rheumatoid factor (RF) by spleen cells of normal adult mice. IgG RF cross-reactive with rabbit IgG was produced in response to immune complexes of TNP-lipopolysaccharide (LPS) with murine IgG anti-TNP antibody in an Fc-specific manner, but not to a mixture of IgG and LPS. Antibody-uncomplexed LPS induced little IgG RF production, but suppressed the subsequent IgG RF response to antibody-complexed LPS, whereas IgM RF was induced by either LPS or antibody-complexed LPS. The IgG RF production followed as rapid a time course as IgM RF production; the rate of IgG RF production reached its maximum soon after a lag period of 1 day and declined after 5 days. Treatment of splenic B cells from BALB/c mice with anti-Ly-1.2 antibody and rabbit complement resulted in a selective reduction of IgM RF production by 90%, with little effect of IgG RF production. These results suggest that IgG RF is derived primarily from CD5- memory B cells which have been developed in normal mice by an unknown mechanism. Unlike the CD5+ precursor cells for IgM RF, these memory cells are unresponsive to polyclonal stimulation by LPS but are activated by simultaneous stimulation by aggregated Fc epitopes and the mitogenic stimulus from LPS.

Animals

IgG isotype and isotype specificity of murine monoclonal IgG rheumatoid factors.

Immune complexes of lipopolysaccharide (LPS) with homologous IgG antibody induces rheumatoid factor (RF) predominantly of the IgG class in normal mice, while LPS alone induces mostly IgM RF directed to homologous IgG1. In this study, IgG monoclonal RFs (mRF) were prepared from hybridomas derived from spleen cells of BALB/c mice which were immunized with complexes of TNP-LPS with anti-TNP mouse IgG and their specificity to mouse IgG subclasses was assessed by analysing dissociation kinetics of the ligands due to RF-specific and non-specific interactions. Of the 19 IgG mRFs (11 IgG1, five IgG2a, one IgG2b and two IgG3 types) tested, 14 were directed to either IgG3 or IgG2b or both, while only one exhibited a significant binding capacity to IgG1. Other mRFs, although reactive to rabbit IgG, exhibited little homophilic activity. None of these mRFs reacted strongly with their own isotypes. The results suggest that the IgG RF producing cells are not direct progenies of the IgG1-directed IgM RF-producing cells but may have developed via a rigorous selection process to eliminate clones that produce self-reactive RF.

Animals

[Detection of cross-reacting protein antigen from urinary tract pathogens by using reversed passive gelatin agglutination test].

The basic studies on detection and titration of the 60-kDa cross-reacting protein antigen (CRPA), which is common to gram-negative rods, by reversed passive gelatin agglutination test with monoclonal and polyclonal antibodies were performed. The gelatin particles sensitized with 500 micrograms/ml of the monoclonal antibody had an ability to react with 200 ng/ml of the purified CRPA for 1.5 h. In contrast, the particles sensitized with 200 micrograms/ml of the polyclonal antibody had an ability to react with 25 ng/ml of the purified CRPA for 1.5 h. In addition, the detection of the 60-kDa CRPA in urinary tract pathogens, which consisted of 60 bacterial strains representing 14 species, was carried out by using the gelatin particles sensitized with 200 micrograms/ml of the polyclonal antibody. The CRPA was detected in all gram-negative rods of urinary tract pathogens, but not in gram-positive cocci. These results suggested that the detection of the 60-kDa CRPA by reversed passive gelatin agglutination test was a rapid and simple method for screening gram-negative bacteriuria.

Agglutination Tests