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Biomedical subjects

F Endo

Publications and source records attributed to F Endo.

At least 163 records · Page 9Linked to original sources
PubMed1987

Screening for late neonatal vitamin K deficiency by acarboxyprothrombin in dried blood spots.

Acarboxyprothrombin (protein induced by vitamin K absence or antagonist-II (PIVKA-II] concentrations in dried blood spots were determined in 19,029 infants at about 1 month of age as an indicator of vitamin K deficiency. We observed 51 cases with raised blood concentrations of PIVKA-II (greater than 4 AU/ml), nine of whom showed very high concentrations (greater than 20 AU/ml). For infants who did not receive vitamin K prophylaxis at birth, the incidence of the PIVKA-II test yielding positive results was significantly higher in those solely breast fed (0.51%) compared with those fed formula milk (0.18%). Among solely breast fed infants, the incidence of a very high result of the PIVKA-II test was 0.14% in those who had not received vitamin K prophylaxis at birth, 0.04% in those who received 2 mg orally, and 0.03% in those who received 2 mg orally plus a further dose of 2-4 mg orally at 7 days. Thus vitamin K prophylaxis at birth did not completely prevent vitamin K deficiency at 1 month. We administered vitamin K therapeutically to all infants whose PIVKA-II test yielded a positive result at 1 month. Only one infant with a positive result developed late neonatal intracranial haemorrhage.

Biomarkers↗
PubMed1987

Altered kinetic properties of the branched-chain alpha-keto acid dehydrogenase complex due to mutation of the beta-subunit of the branched-chain alpha-keto acid decarboxylase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complexes of lymphoblastoid cell lines derived from patients with classical maple syrup urine disease (MSUD) phenotypes were studied in terms of their catalytic functions and analyzed by immunoblotting, using affinity purified anti-bovine BCKDH antibody. Kinetic studies on three cell lines derived from patients with the classical phenotype showed sigmoidal or near sigmoidal kinetics for overall BCKDH activity and a deficiency of the E1 component activity. An immunoblot study revealed a markedly decreased amount of the E1 beta subunit accompanied by weak staining of the E1 alpha subunit. The E2 and E3 component exhibited a cross-reactive peptide. Thus, in at least some patients with MSUD, mutations of the E1 beta subunit might provide an explanation for the altered kinetic properties of the BCKDH complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Immunochemical studies of human prolidase with monoclonal and polyclonal antibodies: absence of the subunit of prolidase in erythrocytes from a patient with prolidase deficiency.

Prolidase was highly purified from human liver and erythrocytes. NaDodSO4/acrylamide gel electrophoresis revealed that these preparations contained a major protein with MW = 56,000. The mass of prolidase was estimated on gel filtration to be MW = 97,000, for both enzyme preparations. A monoclonal antibody was raised against the liver enzyme and a specific antiserum against the erythrocyte enzyme. The monoclonal antibody (EP-2) recognized prolidase from erythrocytes and liver, in equal proportions. The antiserum also recognized the enzyme from erythrocytes and liver. Immunoprecipitation studies with these antibodies suggested only a single species of prolidase in erythrocytes and liver. Using an immobilized monoclonal antibody (EP-2) as an immunoadsorbent, prolidase was partially purified from crude extracts, and the protein of the partially purified enzyme was identified by immunoblotting using antiserum. A protein band with a MW = 56,000 was demonstrated specifically when crude extracts from the liver and erythrocytes were examined using NaDodSO4/acrylamide gel electrophoresis. The subunit protein was absent in erythrocytes from a patient with prolidase deficiency. We propose that the absence of the subunit is one cause of the prolidase deficiency.

Antibodies, Monoclonal↗
PubMed1987

Structural analysis of normal and mutant insulin receptors in fibroblasts cultured from families with leprechaunism.

Leprechaunism is an inherited disorder characterized by insulin resistance and intrauterine growth restriction. In this study we analyze insulin binding and subunit structure of the insulin receptor in dermal fibroblasts cultured from three unrelated families whose probands (Ark-1, Atl, and Minn) were affected by leprechaunism. Cells cultured from all three probands had markedly reduced insulin binding at equilibrium. Fibroblasts cultured from the parents of Ark-1 and Atl had partial and differing degrees of impairment in insulin binding. The structure of the alpha subunit of insulin receptors was analyzed by cross-linking 125I-insulin to plasma membranes. A major band of 350 kilodaltons (kD) (corresponding to the heterotetrameric insulin receptor alpha 2 beta 2) was observed in control and leprechaun fibroblasts. The relative amount of radioactivity cross-linked to plasma membranes reflected the genetic variations seen in insulin binding to intact cells. In reducing gels, 125I-insulin was cross-linked equally to a 250-kD (alpha-alpha dimer) and a 125-kD (alpha monomer) protein in cells from controls, the parents of Ark-1 and Atl, and probands Atl and Minn. By contrast, cells from the Ark-1 proband had diminished cross-linking of alpha-alpha dimers. The ratio of dimer to monomer in cells from controls was 0.93 +/- 0.06, and that in cells from Ark-1 was 0.31 +/- 0.19 (P less than .01). Beta-subunit structure and function was analyzed by studying insulin-enhanced autophosphorylation. Although maximal stimulation of beta-subunit phosphorylation was reduced to 30% in proband Ark-1 fibroblasts, this reduction was quantitatively related to reduced insulin binding. These results indicate that mutations causing severe insulin resistance and defective insulin binding are transmitted with autosomal recessive patterns of inheritance and that heterogeneity exists for these mutations. The mutation in pedigree Ark-1 most likely produces conformational changes in alpha-subunit interaction.

Cells, Cultured↗
PubMed1986

Familial supernumerary non-satellited microchromosome.

A small, non-satellited and metacentric supernumerary chromosome was ascertained in an amniotic fluid cell culture. The supernumerary chromosome was present in four of seven family members karyotyped. Of these four carriers, two were phenotypically normal, while the other two, the fetus and its elder sister, suffered from methylmalonic acidemia. The supernumerary chromosome was apparently stable, being transmitted through three generations without causing mosaicism. It had a single C band, seven G bands and was silver staining negative, a hitherto undescribed situation.

Adult↗
PubMed1986

Influence of the type of feeding on the presence of PIVKA-II in infants.

PIVKA-II levels were studied by a highly sensitive immunological method in two groups of infants, breast-fed and bottle-fed, at the age of 4 days, 1 month, 2 months, and 3 months. PIVKA-II could be demonstrated in 9 infants after the age of 1 month when they were breast-fed. In none of the bottle-fed infants PIVKA-II was present during the same period. This significant difference can probably be explained by the lower vitamin K1 content of human milk compared to commercial formulas. The frequently occurring biochemical deficiency of vitamin K indicates the need of prophylactic administration of vitamin K to all newborns.

Biomarkers↗
PubMed1986

[Penetration of cefotaxime into human bone marrow blood].

Concentrations of cefotaxime (CTX) in bone marrow blood and venous blood were examined with the passage of time in 21 cases which received operations of bone and joint. Concentrations of CTX in bone marrow blood at 30 minutes of a single intravenous administration 2 g each of CTX were found to be 85.2 +/- 24.5 micrograms/ml. Concentration ratio of CTX in bone marrow blood to that in venous blood was reached the peak at 120 minutes after administration. Concentrations of CTX observed were higher than the MIC of CTX against major pathogens responsible for the postoperative infections in orthopaedic field. The CTX, therefore, is expected to have an effective antibiotic in prophylaxis.

Adolescent↗
PubMed1985

Effect of vitamin K administration on acarboxy prothrombin (PIVKA-II) levels in newborns.

PIVKA-II (protein induced by vitamin K absence or antagonist-II) was measured in two groups of newborns, one group being given 5 mg vitamin K at birth and the other untreated. The untreated group had a significantly higher proportion of PIVKA-II positive babies at 3 and 5 days of age than did the treated group. When vitamin K was administered to newborn babies whose normotest levels were less than 30%, it was found that the higher the pre-treatment PIVKA-II levels the greater the response to vitamin K, as monitored by the normotest. Thus PIVKA-II levels might be more useful than a coagulation test, since the low activity of vitamin K dependent coagulation factors sometimes reflects not vitamin K deficiency but impaired production of these factors because of immaturity. The findings support the view that vitamin K given prophylactically at birth will help to prevent neonatal bleeding.

Administration, Oral↗
PubMed1985

Detection of vitamin K deficiency by use of an enzyme-linked immunosorbent assay for circulating abnormal prothrombin.

A monoclonal antibody was raised against an abnormal decarboxylated prothrombin by a cell fusion technique. A cell line which produces an IgG1 murine antibody to the abnormal prothrombin, but not to prothrombin, was selected. Using this antibody we developed an enzyme-linked sandwich immunoassay for the abnormal prothrombin. The detection range was 0.5 X 10(-1) approximately 0.5 X 10(-3) micrograms protein of decarboxylated prothrombin and 0.5 approximately 0.5 X 10(-2) micrograms protein of abnormal prothrombin in vitamin K-deficient subjects. This discrepancy is attributable to a heterogeneity of decarboxylated prothrombin, depending on the number of gamma-carboxyglutamic acid residues. The antibody obtained had a higher affinity to a protein possessing less gamma-carboxyglutamic acid residues. The assay system developed may be useful for the detection of vitamin K deficiency, since a severe deficiency may result in less gamma-carboxyglutamic acid residues in the protein.

1-Carboxyglutamic Acid↗
PubMed1985

Regulation of receptors and digestive activity toward synthesized formyl-chemotactic peptide in human polymorphonuclear leukocytes.

A receptor binding and digestive activity of human polymorphonuclear leukocytes (PMNs) toward formyl-methionyl-leucyl-[3H]phenylalanine (3H-FMLP) was examined with the following results: Up- and down-regulation and recovery of 3H-FMLP binding activity were demonstrated. Both intact PMN and a lysate prepared from them cleaved the carboxyl terminal amino acid (phenylalanine) of 3H-FMLP. The digestive activity decreased as the receptor binding was inhibited by n-ethylmaleimide and 4-chloromercuribenzoate. Little digestive activity was found in the supernatant from PMN stimulated by FMLP. The released phenylalanine was found in the pellet and supernatant of PMNs. Digestive activity with cathepsin A-like characteristics was found in the lysate of PMN. These observations suggest that FMLP is internalized in lysosomes in a receptor-mediated manner and cleaved by the cathepsin A-like enzyme, the free phenylalanine is released extracellularly, and a part of the dissociated receptors with FMLP may return to the surface or to an intracellular receptor pool. Another finding was that the digestive activity of the lysate of cord blood granulocytes was decreased compared with that of adult blood granulocytes. This decrease may explain in part the impaired chemotaxis of cord blood granulocytes.

Cell Membrane↗
PubMed1985

Leprechaunism: an inherited defect in a high-affinity insulin receptor.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,120 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = 240 microU/ml), and the mother was normal. [125I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, 2%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of 2-methylaminoisobutyric acid uptake occurred with half-maximal responses between 25 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of 2-deoxy-D-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [125I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [125I]insulin specifically bound to 125,000 dalton monomeric subunits and 250,000 dalton dimers. In control cells, the ratio of monomer to dimer was approximately one, but significantly fewer dimers were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.

Binding Sites↗
PubMed1984

Establishment of human monoclonal anti-DNA antibody producing cell lines.

We developed a useful method for the establishment of stable cell lines producing human monoclonal anti-DNA antibody by in vitro Epstein-Barr virus infection. The practical limitation for the cloning was overcome by 2 procedures. One was a microculture system using a small number of the culture. Another was enrichment of anti-DNA producing cells at an early stage and prior to the cloning. The combination of these procedures allowed ready derivation of the cell lines secreting monoclonal anti-DNA antibody. Sixteen cell lines were cloned by utilizing colony formation methods in soft agarose. About 14-32 micrograms per ml of IgM with specific antibody activity were obtained in the supernatant of the cells. The antibody reacted with double-stranded and/or single-stranded DNA. These cells have been continuously producing the specific antibody for more than 3 years. We may extend this procedure for obtaining other autoantibodies, such as anti-T cell antibodies.

Animals↗
PubMed1984

Hyperimmunoglobulin-E-associated recurrent infection syndrome accompanied by chemotactic inhibition of polymorphonuclear leukocytes and monocytes.

An 8-yr-old girl with a history of severe recurrent infections including perinephritic, pulmonary, and hepatic abscesses had elevated serum IgE levels. Her serum inhibited chemotaxis of polymorphonuclear leukocytes (PMN) and monocytes. Exchange blood transfusion or plasma exchange at the time of severe infection resulted in normalization of chemotactic activity of PMN shown by the skin window method. Although this effect became negative 1 wk after the treatment, the procedures improved her clinical course. The patient's serum, obtained by exchange blood transfusion, 1) inhibited normal PMN chemotaxis toward cultured supernatant of E. coli, zymosan-activated serum, and formyl methionyl-leucyl-phenylalanine (f . Met-Leu-Phe), a synthetic chemotactic peptide; 2) inhibited monocyte chemotaxis, 3) showed an absence of digestive activity of f . Met-Leu-Phe, 4) was heat stable at 56 degrees C for 30 min and 5) showed an absence of antigenicity of IgE in a partial purified inhibitor with a molecular weight of 30,000-40,000. The inhibitory effect seemed to be reversible.

Bacterial Infections↗
PubMed1984

Structural analysis and subunit interaction of insulin receptor from membranes of cultured embryonic chick heart cells.

In previous studies of cultured embryonic chick heart, insulin hyperpolarized cells and slowed their beat rate through occupancy of a high affinity receptor. In the present studies, we chemically characterize the native structure and subunit interactions of this insulin receptor. A stokes radius of 87 A and an apparent molecular mass of 350,000 daltons were found for membrane proteins specifically cross-linked to [125I] insulin by disuccinimidyl suberate. A primary subunit of 125,000 daltons in dithiothreitol or 115,000 daltons in its absence (alpha-subunit) was heavily cross-linked. A smaller subunit had a size of 90,000 daltons (beta-subunit). This beta-subunit was not readily labeled by [125I]insulin cross-linking, but insulin enhanced 32P incorporation from [gamma-32P]ATP, enabling its visualization. Subunit interaction could be studied, because alkaline conditions produced dissociation of the native 350,000-dalton receptor. This spontaneous dissociation was not a result of proteolysis and was prevented by acid conditions, oxidants, or N-ethylmaleimide. Using alkaline conditions followed by chemical reduction in two-way gels, we directly visualized the native complex of 350,000 daltons dissociating into combinations of subunits of apparent sizes of 290,000 (alpha 2 beta), 220,000 (alpha alpha), and 195,000 (alpha beta) daltons. Dithiothreitol produced combinations of subunits, which differed from alkaline dissociation. In alkaline conditions, the 290,000 (alpha 2 beta) and 220,000 (alpha alpha) dalton combinations predominated, whereas dithiothreitol produced 190,000 (alpha beta)-dalton proteins, which suggested that alpha-S-S-alpha disulfide bonds existed and were susceptible to chemical reductants, while alpha-S-S-beta disulfide bonds were more sensitive to alkaline lysis. We conclude from these observations that the native insulin receptor of embryonic chick heart cell exists on the sarcolemmal membrane as a relatively homogeneous tetramer of nonhomologous subunits in an alpha 2 beta 2 configuration. The alpha-subunits are the primary sites for insulin binding, and a beta-subunit is autophosphorylated. alpha-S-S-alpha and alpha-S-S-beta bonding exist, and these disulfide bonds have different sensitivities to chemical reducing agents and alkaline lysis.

Adenosine Triphosphate↗