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N Yoshida

Publications and source records attributed to N Yoshida.

At least 505 records · Page 28Linked to original sources
PubMed1988

Glycophosphatidylinositol-anchored proteins in metacyclic trypomastigotes of Trypanosoma cruzi.

We searched for the presence of glycophosphatidylinositol (GPI)-anchored proteins in epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi, by treatment of parasite lysates with the GPI-specific phospholipase C of Trypanosoma brucei. Upon treatment, several proteins (70-90 kDa) in metacyclics, but none in epimastigotes, reacted with antibodies to the cross-reacting determinant (CRD), an epitope revealed on the variant surface glycoproteins of T. brucei following removal of the diacylglycerol moiety from their GPI-anchor. Since these T. cruzi metacyclic proteins also lost their original amphiphilicity, as judged by Triton X-114 phase separation, it is very likely that they are linked to the membrane by GPI. One of these proteins is the 90 kDa protein, the major surface protein of G and Tulahuen strains, recognized by the monoclonal antibody 1G7. A variable portion of the 90 kDa molecules was resistant to solubilization by T. brucei lipase. The reasons for this are not clear but susceptibility appeared to increase with the age of the T. cruzi culture. Enzymes that solubilize GPI-anchored proteins were detected in epimastigotes and metacyclics, but the enzymatic activity in these forms was smaller than the activity detected in the same cell numbers of trypomastigotes of T. cruzi originated from infected mammalian cells or from T. brucei bloodstream forms. A preliminary characterization of these activities indicates that at least two classes of enzymes, one of them inhibited by o-phenanthroline, are present in epimastigotes and metacyclics. None of the reagents tested fully inhibited the phospholipases.

Animals↗
PubMed1988

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase).

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.

Amino Acids↗
PubMed1988

Reactivity of stage-specific monoclonal antibody 1G7 with metacyclic trypomastigotes of Trypanosoma cruzi strains: lytic property and 90,000 mol. wt surface antigen polymorphism.

Eleven strains of Trypanosoma cruzi, originating from a variety of vertebrate and invertebrate hosts in distinct geographical regions, were examined for the reactivity of metacyclic stages with the monoclonal antibody 1G7. Trypomastigotes of five strains were susceptible to complement-dependent 1G7-mediated lysis. Higher levels of 1G7 bound to metacyclics of lysis-susceptible strains as compared to lysis-resistant isolates. Excluding Y and CL strains, 1G7 reacted with metacyclics of all T. cruzi isolates by binding to a 90,000 mol. wt surface polypeptide. A 90,000 mol. wt protein lacking the 1G7-specific epitope but immunologically related to the 90,000 mol. wt antigen of other T. cruzi isolates is present in Y and CL strains. The intensity of the 90,000 mol. wt band, detected by surface iodination of metacyclics or in immunoblots using the monoclonal antibody 1G7 or the monospecific antiserum to 90,000 mol. wt protein, varied among different strains and also a discrete variation was observed in its molecular weight. The overall analysis reveals a polymorphism of the 90,000 mol. wt protein, which is ubiquitous among the different T. cruzi isolates.

Animals↗
PubMed1988

Trypanosoma cruzi metacyclic trypomastigotes: neutralization by the stage-specific monoclonal antibody 1G7 and immunogenicity of 90 kD surface antigen.

In this study we have investigated the ability of the stage-specific monoclonal antibody (MoAb) 1G7 to neutralize the infectivity of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain). Mice inoculated with metacyclics pre-incubated with 1G7 or the corresponding Fab fragments were found to develop greatly reduced parasitaemias compared with control animals. Similar results were obtained when mice received 1G7 (0.5 mg/mouse) before challenge with metacyclics. The 90 kD surface protein recognized by MoAb 1G7 was affinity purified and analysed for its immunogenic properties. Immunization of mice with 90 kD antigen induced antibodies that reacted with the native metacyclic surface antigen. The purified antigen was also capable of specifically stimulating the 'in-vitro' proliferation of 90 kD-primed T-cells. Furthermore, in a preliminary experiment in which mice were immunized with the purified polypeptide and challenged with Tulahuen metacyclics, low parasitaemia levels were observed in 60% of animals as opposed to non-immunized controls. The iodinated 90 kD antigen, which has previously been shown to be ubiquitous among T. cruzi strains, was precipitated by different immune sera including various chagasic sera.

Animals↗
PubMed1988

Purification, characterization and amino-acid sequencing of two pancreatic secretory trypsin inhibitors in rat pancreatic juice.

We purified two pancreatic secretory trypsin inhibitors (PSTI-I and PSTI-II) from rat pancreatic juice. PSTI-I consisted of 61 and PSTI-II of 56 amino-acid residues. Their amino-acid sequences were similar (40 out of 56 amino acid residues of PSTI-II being identical with those of PSTI-I), but PSTI-I and PSTI-II appeared to be translation products of different genes. There was no difference in inhibitory properties between PSTI-I and PSTI-II.

Amino Acid Sequence↗
PubMed1988

An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine.

A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma-chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

Arginine↗
PubMed1988

Thrombasthenia with an abnormal platelet membrane glycoprotein IIb of different molecular weight.

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.

Adult↗
PubMed1987

In vivo generation and function of B cells in the presence of a monoclonal anti-IgM antibody: implications for B cell tolerance.

C57BL/6 mice were chronically treated with milligram doses of the noncytotoxic monoclonal anti-mu b antibody MB86 (IgG1, kappa) from birth or from fetal life. The spleens of the manipulated animals contained large numbers (25% as compared to control mice) of B lineage cells which expressed IgMb on the surface after overnight incubation in vitro. The spleens also contained B cells whose surface IgM was unreactive with antibody MB86. A few such cells were immortalized by cell fusion. They included cells secreting mu together with lambda 2 chains which apparently prevent recognition by antibody MB86, and a point mutant in the first constant domain of the mu chain, changing the b to the a allotype. Cells expressing MB86- surface IgM did not selectively expand under MB86 treatment over the first few months of life. Serum Ig levels in the manipulated mice were normal except for IgM which was undetectable in most instances. In some animals low levels of MB86- IgM molecules were produced. At 7 weeks of age, mice treated with MB86 from birth produced normal-size IgG anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) responses with the usual predominance of lambda 1 chain-bearing IgG1 antibodies. At the age of 5-6 months, and also in young mice treated with MB86 from fetal life, the responses were variable and presumably oligoclonal, with a tendency towards the production of antibodies with gamma 3 heavy and lambda 2 or lambda 3 light chains. We interpret these results to mean that B cells hit by antibody MB86 from the time of their generation become unresponsive to T cell-dependent stimulation, but are still able to expand. Occasionally, they escape functional suppression through class switching (to IgG3) upon mitogenic stimulation. At birth, C57BL/6 mice contain a mature B cell population which mediates normal immune responses under MB86 treatment and eventually dies out. Taken as a model of tolerance induction in B cells, the data provide evidence for "tolerant" cells and support the concept of an early phase of sensitivity to tolerance induction in B cell differentiation. The anti-NP response under MB86 treatment differed profoundly from control responses in idiotypic terms, but became normal as the animals recovered from suppression. This may reflect blockade by MB86 of idiotypic selection within the B cell population.

Animals↗
PubMed1987

Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.

Animals↗
PubMed1987

Aromatase.

Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-450 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-450 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-450 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for androstenedione on reconstitution with NADPH-cyt P-450 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-450 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-450 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, 4-14C]androstenedione led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.

Androgens↗