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Biomedical subjects

M Inomata

Publications and source records attributed to M Inomata.

At least 19 recordsLinked to original sources

Slow axonal transport of soluble proteins and calpain in retinal ganglion cells of aged rabbits.

The rate of slow axonal transport of soluble proteins in retinal ganglion cells of the rabbit decreased with approximately 25% in aged (6 years) compared to previous estimates in adult (2 years) animals. Immunobinding of calpain to microtiter plates coated with a monoclonal antibody to mu-calpain was used to isolate labelled axonally transported mu-calpain from the nerve extracts. It was found that the distribution of labelled mu-calpain in the retrobulbar optic pathway was similar to the distribution profile of the slowly migrating phase of soluble proteins.

Aging

Primary anorectal malignant melanoma.

A case of primary anorectal malignant melanoma seen in a 46 year old woman is presented herein. Her most marked symptoms were bloody stools and anal pain. Endoscopic examination indicated a tumor with ulceration but without pigmentation in the anorectal region. Histologic examination of the biopsied specimens showed spindle-shaped cells with atypia proliferating in a bandlike arrangement, as in leiomyosarcoma. An abdominoperineal resection was done and detailed histological examination of the tumor confirmed the nature of the tumor to be malignant melanoma. The postoperative immunochemotherapy consisted of Dimetyl-Triazeno-Imidasole-Carboxamide (DTIC), Amino-Methyl-Pyrimidinyl-Methyl-Chlorethyl-Nitrosourea-Hydrochlori de (ACNU), Vincristine (VCR) and OK-432. The patient has been well without recurrence for fifteen months following her operation through the continuous administration of these agents.

Antineoplastic Agents

Calcium-activated neutral protease (CANP) in normal and dysmyelinating mutant paralytic tremor rabbit myelin.

Calcium-activated neutral protease (CANP) in normal and dysmyelinating mutant, paralytic tremor (PT) rabbit myelin and premyelin fractions was studied using immature (4-5 wk) or adult animals. The enzyme was estimated by determination of its catalytic activity as well as by using immunoblot analysis after SDS-PAGE separation. The presence of two forms of CANP--one activated by calcium in the micromolar concentration (mu CANP) range and the other exhibiting low calcium sensitivity in the millimolar concentration range (m-CANP)--was found in the myelin and premyelin fractions. The developmental pattern of the enzyme activity was different for each of these two enzyme isoforms depending on the fraction studied. The higher activity on CANP (both isoforms) found in PT myelin and premyelin could be related to delayed myelination and/or to the higher turnover rate of already formed myelin. These results suggest complex and specific roles for these isoenzymes during myelin formation as is discussed further in this article. Our results confirm the extensive degradation of myelin basic protein (MBP), proteolipid protein (PLP), and, to a lesser extent, the other myelin proteins by endo- and exogenous CANP. This degradation process was significantly elevated in PT rabbit myelin. Moreover as was shown by two-dimensional gel electrophoresis, calcium-controlled proteolysis in nonmutant rabbits affected the net-charge of MBP in a manner similar to that reported for PT myelin, suggesting the possible involvement of CANP in the generation of charge isomers of MBP.

Animals

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors.

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.

Amino Acid Sequence

Developmental and dietary induction of the 90K subunit of rat intestinal phytase.

The activities of phytase and alkaline phosphatase in the intestine gradually increased in parallel during development of rats, but the 70K and 90K subunits were expressed differentially; only the 70K subunit was detected at birth, whereas the 90K subunit appeared at the weaning period (3 weeks after birth). When rats were forced to wean at 18 days old and fed laboratory chow, the enzyme activity increased markedly and the 90K subunit appeared within 1 day. These findings suggest that weaning is involved in the change in the subunit composition. Increases in the enzyme activity and amount of the 90K subunit were significantly delayed by feeding weanling animals on casein diet, but induced significantly by feeding them on casein diet supplemented with phytate. Thus induction of the 90K subunit seems to be accelerated by intake of phytic acid in the diet. The Km value of the enzyme from suckling rats for phytate was 5.25 mM, while that of adult rats was 0.213 mM. In contrast, the Km value for p-nitrophenyl phosphate (PNPP) was constant during development. The phytase activity of suckling rats did not show a distinct pH-dependence. These findings suggest that the 90K subunit may play some important roles in expressing an efficient phytase activity.

6-Phytase

Y/6 chromosome translocation in a male with triple primary cancers involving the breast.

Cytogenic studies were performed in a 72-year-old male patient with triple primary cancers including breast, skin and lung. Left breast cancer was diagnosed at the age of 46 and he received mastectomy and thoracic irradiation. Squamous cell carcinoma and Bowen's disease were diagnosed from two separated parts of a skin lesion at the age of 70. Small-cell lung cancer was diagnosed 1 year later, and he received chemotherapy and radiotherapy. Chromosome analysis was carried out on both peripheral lymphocyte and skin fibroblast cultures at the age of 72. Out of 30 fibroblast cells karyotyped at the second passage, 7 cells (23%) consistently showed a reciprocal translocation t(Y;6)(q12;p21). The same translocation was found in one of 200 cells from lymphocyte cultures. The findings suggest that the translocation t(Y;6) might be inherent in nature, and that the patient was a mosaic of 46,XY/46,X,t(Y;6)(q12;p21). These results highlight the constitutional chromosomal abnormality as one of the possible high-risk factors for multiple primary cancers.

Breast Neoplasms

Reversal of cisplatin resistance with amphotericin B in a non-small cell lung cancer cell line.

The potentiation of anticancer agents by non-anticancer drugs is one of the possible strategies for overcoming cellular resistance to chemotherapy. In order to overcome cis-diamminedichloroplatinum(II) (CDDP) resistance, we evaluated the sensitizing effect on CDDP-induced cytotoxicity of various non-anticancer agents which might alter membrane transport, by means of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Drugs which have previously been demonstrated to modify multidrug resistance did not show a sensitizing effect to cisplatin. Only amphotericin B (AmB) selectively conquered CDDP resistance in the CDDP-resistant cell line. A drug accumulation study done by the atomic absorption method demonstrated that the accumulation of CDDP in the resistant cell line recovered to the level of the parental cell line after treatment with AmB. Thus, AmB might overcome CDDP resistance by increasing the accumulation of CDDP.

Amphotericin B

Ability of young children to button and unbutton clothes.

From 2 years of age, children enjoy trying to button and unbutton their jacket. The clothing of the children at this stage should have the form which fits their motor skills so as to develop their interest in buttons and to help their study in manipulating buttons. We observed processes of dressing and actions of buttoning and unbuttoning at the nursery school, for children in the 3.3-5.9 year range. Also, we experimented about dressing mainly for children in 2 year range to observe the process of manipulating buttons. As factors of experiment, we took two buttonhole directions (vertical and across) and three sizes of buttons (1, 2, 3 cm). The direction of buttonholes was significant at the 5% level. We recognized that buttonholes in the vertical direction were easier for children to button. We came to the following conclusion: for an open front of children's clothing, buttons having a 2 cm diameter and buttonholes in the vertical direction were the best.

Age Factors

Binding sites for calcium-activated neutral protease on erythrocyte membranes are not membrane phospholipids.

In order to explore the binding sites for calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) on the inner surface of human erythrocyte membranes, we analyzed the binding of muCANP to two kinds of membranes modified by treatment with phospholipase C or Triton X-100. Binding analyses were performed using an immunoblot technique. The amount of muCANP bound to phospholipase C-treated inside-out vesicles was essentially the same as that bound to untreated inside-out vesicles. It was also observed that muCANP binds to Triton X-100-treated membranes, in which most of the integral proteins and glycerophospholipids are removed while the lining proteins remain intact. In both types of modified membrane, the bound muCANP was rapdily converted to an active form by autolysis at physiological free Ca2+ concentrations. These results indicate that the binding sites for muCANP on the inner surface of erythrocyte membranes consist of components other than membrane phospholipids. In addition, it is suggested that one of the binding sites for muCANP is some lining protein.

Animals

Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease.

The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.

Animals

Fragmentation of an endogenous inhibitor upon complex formation with high- and low-Ca2+-requiring forms of calcium-activated neutral proteases.

The interaction of an endogenous inhibitor for the calcium-activated neutral protease (CANP or calpain EC 3.4.22.17) with CANP was examined by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and gel filtration. Fragmentation of the inhibitor (Mr 110K) by mCANP, a high-Ca2+-requiring form, was shown only in the presence of Ca2+ ions of millimolar order, with decreased inhibitor activity recovered from gel extracts in the 110-kDa area. This fragmentation took place even when the inhibitor could completely inhibit the caseinolytic activity of mCANP. The fragmented inhibitor retained considerable inhibitor activity after the CANP-inhibitor complex was dissociated by the addition of EDTA, and 69% of the initial activity was recovered from the mixture reacted with excess mCANP lacking the 110-kDa band. A C-terminal fragment of CANP inhibitor produced in Escherichia coli (Mr 40K) was also hydrolyzed by mCANP in the presence of Ca2+. The interaction of both forms of the inhibitor with mu CANP, a low-Ca2+-requiring form, led to the same phenomena in the presence of micromolar levels of Ca2+. CANP inhibitor could not completely inhibit the autolysis of mCANP and mu CANP, indicating that these were intramolecular events. Gel filtration analysis revealed that the mass of the smallest fragment with inhibitor activity was about 15,000 daltons. These results suggest that CANP inhibitor may act in the manner of a suicide substrate.

Animals

[Evaluation of a modified latex agglutination inhibitory reaction method for the quantitation of urinary estriol in pregnant women].

This study evaluated a modified latex agglutination inhibitory reaction method for the quantitation of urinary estriol in women during normal and abnormal pregnancy. The urinary levels of estriol in pregnant women measured by this method correlated with urinary and serum levels measured by radioimmunoassay. The urinary level of estriol rose during pregnancy and reached a peak at the time of delivery, after which it returned to the control level in a few days. In contrast, the urinary level of estriol in women with abnormal pregnancy did not rise during pregnancy. This method is useful to detect impaired function of the placenta in pregnant women.

Estriol

Activation mechanism of calcium-activated neutral protease. Evidence for the existence of intramolecular and intermolecular autolyses.

The activation mechanism through limited autolysis of a calcium-activated neutral protease (CANP) with a high sensitivity to calcium ions (microCANP) was analyzed. The rate of autolysis was dependent on microCANP concentration. The reaction was inhibited by high concentrations of digestible substrates but not by a nondigestible substrate. Incubation of microCANP inactivated by N-ethylmaleimide with a small amount of activated microCANP caused the degradation of the former in a manner similar to the autolysis of native microCANP. Immobilized microCANP bound to an anti-microCANP immunoglobulin G column autolyzed on addition of calcium ions. These results show that activation of microCANP through limited autolysis involves both intramolecular and intermolecular reactions.

Animals

Chemosensitivity profiles of primary and cultured human retinoblastoma cells in a human tumor clonogenic assay.

The drug sensitivity of retinoblastoma cells obtained from 14 fresh primary materials (13 from enucleation and 1 from autopsy) and 2 cultured lines (Y-79 and WERI-Rb1) was determined using the human tumor clonogenic assay developed by Hamburger and Salmon. Components of the conventional soft agar medium were slightly modified to make them suitable for growing primary retinoblastoma cells. More than 5 colonies were formed by all 14 primary samples tested from the 500 X 10(3) cells plated. More than 30 colonies per dish were formed from the 13 samples, with a median plating efficiency of 0.033% (0.005-0.400), and these were used in the in vitro measurements of drug chemosensitivities. They showed homogeneous sensitivity to the representative alkylating agent L-phenylalanine mustard; 13 out of 14 showed a decrease in the colony formation of more than 70%. The other drugs which were effective (more than 70% colony inhibition) against the primary retinoblastoma cells were: doxorubicin (7 out of 13), mitomycin C (7 out of 13), actinomycin D (4 out of 13), cis-diamminedichloroplatinum(II) (3 out of 13), nimustine (1 out of 13), and peplomycin (1 out of 13). Vincristine, bleomycin, 5-fluorouracil, methotrexate, decarbazine, and cytosine arabinoside were not effective. When the chemosensitivity of retinoblastoma cells of the two established cell lines was examined by the same method, only L-phenylalanine mustard was effective against Y-79, and no drug was effective against WERI-Rb1.

Antineoplastic Agents

Autolytic activation of calcium-activated neutral protease.

Degradation of vimentin by native low calcium ion-requiring protease (mu CANP) was compared to that by autodigested mu CANP. On activation with 5 mM barium ions, a lag time was observed for the case of native mu CANP. This provides direct evidence that native mu CANP is inactive as a protease and must be autolyzed to be activated. Most of the protease activity can be accounted for by autodigested mu CANP with a 76 K polypeptide but another species with 50 K polypeptide may also be active.

Animals

Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity.

Fifteen hybridomas secreting antibodies against calcium-activated neutral protease (CANP), especially those for rabbit muscle mCANP with low calcium sensitivity, have been produced by the cell fusion technique. Eight of the monoclonal antibodies belong to the class IgG1, one to the class IgG2a, and six to the class IgG2b. The antibodies from these clones were characterized with regard to their relative binding affinities to the large subunits (80K) and the small subunits (30K) of mCANP as well as mu CANP, which is another type of CANP with high calcium sensitivity. Fourteen antibodies bound only to the 80K subunit of mCANP and one antibody bound to the 80K subunit of both mCANP and mu CANP. These antibodies recognized rat mCANP but not chicken CANP, with the exception of one antibody. Examination of the effects of these antibodies on the enzyme activity of mCANP showed that six antibodies partially inhibited the enzyme activity and the others were noninhibitory. These monoclonal antibodies should be useful for analyzing the fine structure of CANPs and the mechanism of the activation of mCANP, and also for determining the intracellular localization of mCANP.

Animals