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Biomedical subjects

J Sawada

Publications and source records attributed to J Sawada.

At least 19 recordsLinked to original sources
PubMed1992

Identification of metallothionein in cultured cells by immunoblotting and immunofluorescence using a new monoclonal antibody.

A hybridoma clone (MT45-5-3) producing an IgG-class monoclonal antibody specific for metallothioneins (MTs) was established. The monoclonal antibody (MT45) cross-reacted with mouse, rat and rabbit Cd(2+)-induced MTs 1 and 2 and Zn(2+)-induced MT 2 as assessed by enzyme-linked immunosorbent assay. When the antibody was used to detect the MTs transferred to nylon membranes after SDS-polyacrylamide gel electrophoresis, the antibody reacted with cultured human cell MTs as well as rat, mouse and rabbit MTs 1 and 2 even after carboxymethylation. The antibody could be used for the indirect immunofluorescence test for Cd(2+)-induced MTs in cultured human and mouse cells.

Animals↗
PubMed1992

Amino acids and peptides. XXXIII. Synthesis of N-terminal epitope peptides of mammalian metallothioneins (MTs).

In order to determine the fine structure of the mammalian metallothionein (MT) epitope to a monoclonal anti-rat Zn-MT-II antibody (MT 189-14-7), N-terminal peptides of various lengths of mammalian metallothioneins (MTs) were synthesized by a conventional solution method using the newly developed beta-2-adamantylaspartate, and their immunological properties were examined. It was found that the N-terminal acetyl group was indispensable for the reaction with the monoclonal antibody and the N-terminally acetylated pentapeptide, Ac-Met-Asp-Pro-Asn-Cys-OH, was the smallest peptide which exhibited a significant reactivity with the antibody.

Amino Acid Sequence↗
PubMed1991

A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay.

A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 x 10(9) M-1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6 beta- and 7 beta-hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and DL-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay-1 (0.2 ng ml-1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.

Antibodies, Monoclonal↗
PubMed1991

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.

17-alpha-Hydroxyprogesterone↗
PubMed1991

[A case of lead neuropathy--importance of subclinical entrapment of nerves in lead neuropathy].

The patient was a left handed 25-year-old man who had worked in a vinyl chloride resin factory since July 1987 using lead stearate as a stabilizer. During the two years preceding hospitalization, he had been admitted three times for colicky abdominal pain with constipation and nausea. Anemia and proteinuria without causative diseases were pointed out. Because of progressive muscle weakness of hands which began since January 1989, he was admitted to our neurologic clinic on October 2 1989. Neurological examination showed severe atrophy of both small hand muscles without sensory disturbances. Left hand was predominantly affected, especially in interossei muscles. Laboratory findings are as follows; RBC 3.25 million/cu.mm: Hb 9.7 g/dl:blood lead concentration 100 micrograms/dl: urinary coproporphyrin 4503 micrograms/l: urinary delta-ALA 138 mg/l: and urinary lead excretion after 1 g CaEDTA infusion: 3938 micrograms/day. Electromyography of extensor carpi radialis, flexor carpi ulnaris and opponens showed increased polyphasic, long duration MUPs. Electromyography of dorsal interossei showed fibrillation. Nerve conduction study showed mildly decreased motor and sensory conduction velocities and markedly reduced amplitude of compound muscle action potentials (CAMPs). Investigation by inching method revealed conduction block of left ulnar nerve across elbow. After CaEDTA chelating therapy, 1 g once a week, steady improvement of muscular weakness was observed within a few months. Serial nerve condition studies revealed constant recovery of nerve conduction velocities, amplitude of CMAPs and diminution of conduction block across the left elbow. Conduction block across left elbow without sensory disturbances implies that subclinical cubital tunnel syndrome might have existed in this patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Antigenic determinants on rat metallothionein: fine epitope mapping for a murine monoclonal antibody and rabbit polyclonal antisera.

In order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.

Amino Acid Sequence↗
PubMed1990

Production and characterization of monoclonal and polyclonal antibodies against digoxin.

Three hybridoma cell lines (DIG 64. 2B. 5, DIG 104. H10.1 and DIG 222. 4D. 5) producing monoclonal antibodies against digoxin were established, and the properties of these monoclonal antibodies were characterized and compared with three polyclonal rabbit anti-digoxin antisera. Both polyclonal and monoclonal antibodies gave association constants ranging from 10(9) to 10(10) (M-1). The monoclonal antibodies were of the IgG1(kappa) or IgG2b(kappa) subclass. Cross-reactivities of these monoclonal antibodies and polyclonal antisera with various related cardenolides and their aglycones were determined by competitive radioimmunoassay. The monoclonal antibodies were specific for digoxigenin-containing glycosides such as lanatoside C and deslanoside, but not for digitoxigenin-containing glycosides. On the other hand, the specificities of the polyclonal antisera were less strict than the monoclonal antibodies and relatively cross-reactive with digitoxin. It was shown that the RIA for digoxin using DIG 64. 2B. 5 is sensitive enough to detect 0.2-0.3 nM (30-60 pg/tube) of digoxigenin-containing cardenolides.

Animals↗
PubMed1989

[Usefulness and limitations of transesophageal echocardiography in anesthetic practice].

Transesophageal echocardiography (TEE) has been used as a monitor of cardiovascular function and as a diagnostic tool in anesthetic practice. TEE is the only available monitor to detect anatomical abnormalities such as of wall motion as well as valvular abnormalities. Doppler TEE has wider diagnostic functions. TEE is a very sensitive monitor to detect myocardial ischemia by recognizing wall motion abnormalities and loss of systolic wall thickening. Preload defined by left ventricular end-diastolic volume may not correlate with left ventricular end-diastolic pressure or pulmonary capillary wedge pressure (PCWP) when left ventricular compliance changes such as after coronary artery surgery. PCWP can be misleading when transmural pressure across cardiac chambers are undetermined such as in patients with cardiac tamponade or those on high positive end-expiratory pressure. In these situations, TEE is a powerful tool to link physiologic parameters and anatomy. TEE is also a very sensitive monitor to diagnose air embolism during cardiac surgery and neurosurgery in sitting position. There are, however, several shortcomings such as its cost, "too much sensitivity", requirement of some experience, interobserver variability, and so on. The computer-assisted on-line analysis would greatly augment usefulness of TEE. When these shortcomings are overcome, TEE would be one of the most important monitors in anesthetic practice.

Anesthesiology↗
PubMed1988

A murine monoclonal anti-metallothionein autoantibody recognizes a chemically synthesized amino-terminal heptapeptide common to various animal metallothioneins.

A murine hybridoma clone (MT 189-14-7) producing an IgM-class monoclonal antibody specific for metallothionein (MT) was produced with rat Zn-MT 2-keyhole limpet hemocyanin conjugate as an immunogen. The reactivity of the monoclonal antibody with MTs obtained from various animal species was determined by the competitive radioimmunoassay. The MT 189-14-7 antibody bound equally to various MTs 1 and 2 including mouse MTs 1 and 2. This indicates that the antibody is a murine autoantibody reactive with various animal MTs and recognizes a common structure of the various MTs. Subsequently, the location of the antigenic determinant recognized by the MT 189-14-7 antibody was determined by using various synthetic human MT 2 peptides. Among the synthetic peptides examined, both the amino-terminal beta-domain, Ac-(hMT 2 1-29)-OH, and an amino-terminal heptapeptide, Ac-(hMT 2 1-7)-OH, but not the carboxy-terminal alpha-domain peptides, H-(hMT 2 29-35)-OH and H-(hMT 2 30-61)-OH, showed inhibitory activities slightly higher than the native MTs in the competitive radioimmunoassay. These results demonstrate that the MT 189-14-7 autoantibody recognizes the epitope located within the amino-terminal heptapeptide common to various MTs.

Animals↗
PubMed1988

Production and characterization of high-affinity monoclonal antibodies against morphine.

Twelve hybridoma cell lines producing MAbs against morphine were established by using morphine hemisuccinate-conjugated bovine serum albumin as an immunogen. The MAbs belonged to the IgG1 subclass with kappa- or lambda-chains. The association constants of the antibodies ranged from 4.6 x 10(8) to 4.7 x 10(10) (M-1). These antibodies revealed slightly different cross-reactivities with various agonistic opiates and antagonists. In general, the antibodies were strongly cross-reactive with the opiate agonists, codeine, ethylmorphine, dihydromorphine and dihydrocodeine, while their cross-reactivities with norcodeine and the opiate antagonists, naloxone and naltrexone, were weak. The cross-reactivities with dihydromorphinone, dihydrocodeinone, naloxone, naltrexone, dextromethorphan and homatropine varied from clone to clone. Interestingly, certain MAbs displayed weak but significant cross-reactivities with the synthetic opiate, meperidine. However, none of the antibodies was cross-reactive with the opioid peptides, beta-endorphin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalinamide. Radioimmunoassay for morphine using one of the antibodies (MOR 131.5.13) was shown to be sufficiently sensitive (IC50 = 0.1 nM) for the purposes of forensic analysis of morphine. This set of monoclonal anti-opiate antibodies is assumed to be suitable for analyzing the structure-function relationship in the hapten-antibody interaction, since the antibodies revealed similar but not identical cross-reactivities with various morphine related compounds.

Animals↗
PubMed1987

Production and characterization of monoclonal antibodies to 17 alpha-hydroxyprogesterone.

Hybridoma clones producing antibodies to 17 alpha-hydroxyprogesterone (17-OHP) were established by using a 17-OHP-bovine serum albumin conjugate as an immunogen. Six representative IgG-class monoclonal antibodies of high affinity (10(8)-10(9) M-1) showed differential reactivities with several structurally related steroids. Two enzyme immunoassay (EIA) systems (fluorescence EIA and micro-EIA) for 17-OHP using OHP 4B2.2.3, which showed the lowest cross-reactivity with other steroids, were established. The micro-EIA system was shown to be applicable to the mass-screening of congenital adrenal hyperplasia.

17-alpha-Hydroxyprogesterone↗
PubMed1987

Roles of IL-2 and antigen in the later stages of the primary antibody response.

Spleen cells, obtained 2-5 days after in vivo priming with sheep erythrocytes (SRBC), were cultured to determine the presence of plaque-forming cell (PFC) precursors capable of developing into mature PFC under the influence of various stimulants. Lipopolysaccharide (LPS), added together with SRBC at the initiation of a 48-hr in vitro culture, enhanced the PFC response of primed spleen cells. In vivo priming for a minimum of 3 days was required, and maximal numbers of PFC were obtained from spleen cells primed for 4 days. Depletion of T lymphocytes from Day 3-primed spleen cells abrogated LPS-mediated enhancement, and addition of concanavalin A supernatants to the T-cell depleted system restored the enhancement, suggesting that LPS action required co-operation with a product(s) of activated T cells. Addition of various interleukin-2 preparations including recombinant human IL-2 to the system restored the LPS-mediated enhancement. The response of Day 3 cells from which T cells were eliminated as vigorously as possible was similarly restored by the addition of IL-2, LPS and antigen, suggesting that IL-2 reacts directly with PFC precursors that have developed IL-2 receptors. LPS-mediated enhancement, in the presence or absence of T cells, was also markedly dependent on the presence of SRBC during in vitro culture. These data suggest that, in co-operation with IL-2 and other co-factors, antigen plays a significant role in driving the later stages of differentiation and/or division of PFC precursors to mature PFC.

Animals↗
PubMed1986

Comparison of methods for freezing interleukin-dependent murine cell lines.

Various methods for freezing several interleukin-dependent murine cell lines have been compared and were shown to differ in their efficacy from line to line. An interleukin-2-dependent cell line, T572.C5, which was more sensitive to dimethylsulfoxide (DMSO) than the other cell lines, was the most difficult to freeze in a viable state. In order to define better conditions for freezing, the effects of the changes in freezing rates and concentrations of DMSO and of the addition of Ficoll were determined. The optimal freezing rates and DMSO concentrations varied from line to line. The addition of Ficoll increased the viability in some, but not all, cell lines, depending on the freezing conditions used. Thus, the optimal conditions for freezing interleukin-dependent cell lines varied from line to line. In general, the direct freezing procedure was better than the styrofoam-box freezing method. Although programmed freezing under direct temperature control was the most suitable among the freezing procedures examined, unprogrammed procedures also were of practical use.

Animals↗
PubMed1986

An immunological study of a Pb-thionein like protein in rat liver.

Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-thionein like protein (Pb-BP) and Zn-thionein in the liver. To determine of the Pb-BP is a species of metallothionein, immunological properties of this protein were investigated. The results indicate that the Pb-BP is cross-reactive with the antibody against rat Zn-thionein II, strongly suggesting that the Pb-BP is a metallothionein.

Animals↗
PubMed1986

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies.

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.

Animals↗