Biomedical subjects
Publications and source records attributed to T Funakoshi.
We report four cases of acute subdural hematoma (ASDH) in which the hematoma rapidly resolved. Three of the four hematomas were traumatic. The outcome was good in three cases and fair in one. Spontaneous resolution of ASDH is rare.
Prolylendopeptidase activity was increasingly depressed with time from 6 to 24 hr after the start of sensitization in the delayed hypersensitive guinea pig skin lesion induced by bovine gamma-globulin as an antigen. The remarkably depressed activity of the enzyme in the violently inflamed skin began to be restored slowly 48 hr after sensitization, and its activity was ultimately recovered to the original level by 504 hr after a single sensitization in vivo. Depression of the enzymatic activity is caused by a novel prolyendopeptidase inhibitor, whose amino acid composition is 7 Glu, 1 Ser, 2 Gly, 1 Ala, 2 Pro, and 1 Val, generated by inflammation.
Maternal cigarette smoking has been associated with some complications of pregnancy, including low birth weight and increased morbidity. Recently, it has been reported that maternal passive smoking also affects the fetal environment and causes fetal growth disturbance. In this study, we aimed to investigate the effects of maternal passive smoking on pregnant women and their fetuses by measuring cotinine concentrations in maternal urine and umbilical cord blood. The results were as follows: 1) Among 259 pregnant women, 17 cases (6.6%) were active smokers. The women who were not aware of passive smoking at all, were only 39 cases (15.1%). More than 80% of the pregnant women smoked either passively or actively each day. 2) Cotinine concentrations in both maternal urine and umbilical cord blood increased with the increase in passive smoking. Those in maternal serum, however, did not correlate with the increase in passive smoking. 3) The relative birth weight (R.B.W.) of the newborn infants delivered by the mothers whose cotinine concentration was more than 9.0ng/ml (This value represented the mean +1.5SD of the cotinine concentration in the urine from the mother who did not passively or actively smoke) was significantly lower than that of the mothers whose cotinine concentration was less than 9.0ng/ml. It is concluded that the measurement of the cotinine concentration in maternal urine or umbilical cord blood is very useful in estimating the effects of passive smoking on pregnant women. And passive smoking as well as active smoking also has a harmful effect on the fetal growth mechanism.
Previously we isolated and characterized a placental anticoagulant protein (PAP or PAP-I), which is a Ca2+-dependent phospholipid binding protein [Funakoshi et al. (1987) Biochemistry 26, 5572] and a member of the lipocortin family [Funakoshi et al. (1987) Biochemistry 26, 8087]. In this study, three additional anticoagulant proteins (PAP-II, PAP-III, and PAP-IV) were simultaneously isolated from human placental homogenates prepared in the presence of 5 mM ethylenediaminetetraacetic acid. The isoelectric points of PAP-I, PAP-II, PAP-III, and PAP-IV were 4.8, 6.1, 5.9, and 8.1, respectively, and their apparent molecular weights were 32,000, 33,000, 34,000, and 34,500, respectively. Amino acid sequences of cyanogen bromide fragments of these proteins showed that PAP-III was a previously unrecognized member of the lipocortin family, while PAP-II was probably the human homologue of porcine protein II and PAP-IV was a derivative of lipocortin II truncated near the amino terminus. Comparative studies showed that all four proteins inhibited blood clotting and phospholipase A2 activity with potencies consistent with their measured relative affinities for anionic phospholipid vesicles. However, PAP-IV bound to phospholipid vesicles approximately 160-fold more weakly than PAP-I, while PAP-II and PAP-III bound only 2-fold and 3-fold more weakly. These results increase to six the number of lipocortin-like proteins known to exist in human placenta. The observed differences in phospholipid binding may indicate functional differences among the members of the lipocortin family despite their considerable structural similarities.
Reaction of equine alpha 2-macroglobulin (alpha 2M) with methylamine caused generation of 3.7 mol of thiol groups per mole of the protein, and the second-order rate constant of the generation was calculated to be 3.5 M-1 s-1. The inhibitory profile of caseinolytic activity of trypsin indicated that one molecule of equine alpha 2M inhibited two molecules of trypsin, similar to human alpha 2M. The methylamine-treated equine alpha 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human alpha 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine alpha 2M is substantially unperturbed by cleavage of the thiol ester bonds and that the intact thiol ester bonds per se are not essential for the ability of equine alpha 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine alpha 2M showed that this treatment caused only a small change in conformation of the protein. Reaction of the methylamine-treated protein with trypsin induced appreciable changes in the spectra, indicating a large change in conformation of the protein. These findings were consistent with the results obtained by electrophoresis: The band of methylamine-treated equine alpha 2M showed indistinguishable mobility from that of the unmodified protein, indicating that no appreciable change in conformation occurred, and distinctly different mobility from that of the unmodified or methylamine-treated equine alpha 2M when each had reacted with trypsin.
The primary structure of human placental anticoagulant protein was determined by a combination of amino acid and nucleotide sequencing techniques. The carboxymethylated protein was digested with cyanogen bromide, and the resulting peptides were separated by gel filtration and high-performance liquid chromatography. A total of 239 out of 319 amino acid residues were identified from 7 cyanogen bromide fragments. A full-length cDNA clone encoding placental anticoagulant protein was isolated from a human placenta cDNA library. This clone was 1.6 kilobases long and contained a translation initiation site coding for methionine, 957 nucleotides encoding for the mature protein, a stop codon, a poly(A) recognition site, and a poly(A) tail. Analysis of the tryptic-blocked peptide that originated from the NH2-terminus of the protein showed that the terminal methionine was removed and the adjacent alanine residue was acetylated by posttranslational events. Placental anticoagulant protein is composed of 319 amino acids with acetylalanine as the NH2-terminus and has a high degree of sequence identity with lipocortins I and II. It contains four internal repeats, each including a sequence corresponding to a putative Ca2+-dependent phospholipid binding site. Placental anticoagulant protein is a member of the lipocortin/calpactin family.
Previous studies indicated that human placental anticoagulant protein, a member of the lipocortin family, prolonged the clotting time of normal plasma when clotting was induced by brain thromboplastin or by kaolin in the presence of cephalin and calcium. Using a two-stage amidolytic assay to assess factor X activation and a tritiated peptide release assay to assess factor IX activation, we have examined the ability of purified preparations of placental anticoagulant protein (Mr = 36.5 kDa) to inhibit the activation of either factor X or factor IX by a complex of human factor VIIa-tissue factor. Placental anticoagulant protein markedly inhibits factor X and factor IX activation by factor VIIa-tissue factor in a non-competitive manner with Ki values of 40 nM and 70 nM, respectively. Placental anticoagulant protein had no effect on factor Xa amidolytic activity, and its inhibitory activity was not diminished by prior incubation with antibody raised against partially purified plasma extrinsic pathway inhibitor. Binding of placental anticoagulant protein to phospholipid vesicles, crude tissue factor and purified, relipidated human brain tissue factor apoprotein was observed only in the presence of calcium ions. These results indicate that placental anticoagulant protein is a potent factor VIIa-tissue factor inhibitor and suggests that its mechanism of action involves binding to the phospholipid portion of the tissue factor lipoprotein.
An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.
The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.
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NH2-terminal myristyl peptides in mixtures of other peptides having the NH2-terminal blocked with acetyl, formyl or pyroglutamyl groups were selectively determined by high-performance liquid chromatography using a poly(vinyl alcohol) resin column eluted with 50 mM sodium hydrogen carbonate, pH 8.3, and a linear gradient of acetonitrile. This method was applied to the analysis of an NH2-terminal blocked peptide from a peptic digest of the catalytic subunit of adenosine 3':5'-phosphate-dependent protein kinase type II from bovine heart, showing that the peptide has a sequence myristyl-Gly-Asn-Ala.
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The phosphorylation sites of the myelin basic protein from bovine brain were determined after phosphorylation with a cyclic 3':5'-phosphate-dependent protein kinase from the same source. Three phosphorylated peptides were selectively and rapidly separated, before and after dephosphorylation, by reversed-phase high-performance liquid chromatography on a styrene 250 column under alkaline conditions. Partial sequencing of the peptides by automated Edman degradation revealed that the serine-115 residue located in the main encephalitogenic determinant of the protein was a phosphorylation site, in addition to the two phosphorylation sites established (threonine-34 and serine-55).
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