[Induction of long-term remission for the first onset of severe enterocolitis Crohn's disease treated by leukocytapheresis alone].
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Adult↗
Biomedical subjects
M Kaneda
Publications and source records attributed to M Kaneda.
A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease.
A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2--forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558, of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.
Adolescent↗
Cucumisin like protease from the sarcocarp of Benincasa hispida var. Ryukyu.
A protease has been purified from the sarcocarp of Benincasa hispida (Thunb.) Cogn. var. Ryukyu by two steps of chromatography. Its M(r) was estimated by SDS-PAGE to be about 67,000. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but not by EDTA and cysteine protease inhibitors. The substrate having alanine at the position of P1 was the best among the Ala-Ala-Pro-X-pNAs (X = Ala, Lys, Phe, Glu, and diaminopropionic acid (Dap)). The N-terminal sequence of the first 33 residues was determined and 25 of the residues agreed with that of cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicated that the B. hispida protease is a cucumisin like serine protease.
Amino Acid Sequence↗
Oral teratogenicity studies of methyl bromide in rats and rabbits.
Teratogenicity studies of methyl bromide, a widely used fumigant, were conducted in rats and rabbits. Methyl bromide was dissolved in corn oil and administered orally to groups of 24 copulated female Crj:CD (SD) rats at dose levels of 0 (corn oil), 3, 10 or 30 mg/kg/day on days 6-15 of gestation and to groups of 18 artificially inseminated female Kbl:JW rabbits at 0, 1, 3 or 10 mg/kg/day on days 6-18 of gestation. Maternal rats and rabbits were euthanized on respective days 20 and 27 of gestation. Foetuses were examined for survival, growth and teratological alterations. Maternal toxicity was evident in the high-dose groups for both species. In these groups, maternal body weight gains and food consumption were significantly decreased during the dosing and post-dosing periods. Necropsy of maternal rats also revealed erosive lesions in the stomach and the surrounding organs. However, no treatment-related adverse effects were found in foetuses of the treated groups for both rat and rabbit studies. These results led to the conclusion that methyl bromide was not foetotoxic or teratogenic to rat and rabbit foetuses up to dose levels of 30 and 10 mg/kg/day, respectively, at which maternal toxicity was evident for both species.
Abnormalities, Drug-Induced↗
Spectrophotometric determination of neutrophil cytochrome b558 of chronic granulomatous disease.
BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (O2-) by the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b558 (cyt b558), which is one of the oxidase components. Diagnosis of CGD has been performed by the assay of the O2- forming activity, immunological determination of defects in the oxidase components, and or spectrophotometry of cyt b558. However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b558. This report presents an accurate method for the spectrophotometric analysis of cyt b558 in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO-bubbling method. METHODS AND RESULTS: The difference of the reduced minus oxidized cyt b558 spectrum was measured under no interference from oxy Hb at the alpha and beta bands and differentiated as d[delta A]/d lambda (lambda = wavelength) to obtain further evidence for the defects of the cyt b558 heme spectrum. The interference from CO-insensitive met Hb was eliminated by subtracting the absorption peak at the Soret (gamma) band of the contaminating met Hb, which was estimated from the CO-treated and untreated spectra of the same, hemolyzed sample. CONCLUSIONS: This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt b558 with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.
Cytochrome b Group↗
Comparison of phytolacain G, a cysteine protease from fruit of Phytolacca americana, with phytolacain R.
The enzymatic properties of phytolacain G, a protease isolated from green fruit of pokeweed, were compared with those of phytolacain R, a protease obtained from ripe fruit. The optimum pH of phytolacain G was 7.5-8.0 at 37 degrees C using casein as the substrate. The enzyme was strongly inhibited by iodoacetic acid and p-chloromercuribenzoic acid, but not by diisopropyl fluorophosphate or EDTA. These results indicated that phytolacain G was a cysteine protease, like phytolacain R. Nine sites of oxidized insulin B-chain were cleaved by phytolacain G during 20 h of hydrolysis. The six sites cleaved by phytolacain G were also cleaved by phytolacain R. The substrate specificity of phytolacain G was broad, but the preference for hydrophobic residues at the P2 position was similar to the substrate specificity of papain. The amino-terminal sequence of phytolacain G was not identical with that of phytolacain R; however, the amino acid residues conserved in the papain family were also conserved in this enzyme.
Amino Acid Sequence↗
A cysteine protease from young stems of asparagus: isolation, properties, and substrate specificity.
A protease was purified from the growing point of asparagus, Asparagus officinalis, using a cystatin-Sepharose column. The asparagus protease is the first protease isolated from the growing point of a plant tissue and from Liliaceae. Its molecular mass was estimated to be 28 kDa by SDS-PAGE. The optimum pH of the enzyme was 7 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by monoiodoacetic acid, but not by diisopropylfluorophosphate, suggesting that it is a cysteine protease. Asparagus protease had broad specificity on the hydrolysis with oxidized B-chain of insulin as a substrate. However, for the P2 position of the cleavage site, the large hydrophobic side chains of amino acid residues such as Phe, Val, and Leu were considerably preferred. Asparagus protease was similar to papain about specificity at the P2 position. The N-terminal sequence of the first 12 residues was identified and 8 residues among them agreed with that of papain, accompanying the addition of one residue (Ala) to that of papain.
Anilides↗
[Evaluation of a newly developed broth microdilution test method to determine minimum inhibitory concentrations (MICs) of antimicrobial agents for mycobacteria].
We developed a new broth microdilution antimycobacterial susceptibility test for determination of minimum inhibitory concentration (MICs) using an air-dried microplate containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight agents included were streptomycin (SM), isoniazid (INH), rifampicin (RFP), ethambutol (EB), kanamycin (KM), levofloxacin (LVFX), sparfloxacin (SPFX) and clarithromycin (CAM). Serial dilutions of the agents (128 micrograms/ml to 0.125 micrograms/ml) were prepared in microplates, and were reconstituted by inoculation of 0.2ml of cell suspensions (approximately 3 x 10(5) cells/ml). The test plates were incubated at 36 degrees C in 5% CO2, and the growth endpoints were read visually after 5-day, 7-day and 10-day incubations. Four ATCC reference strains, Mycobacterium tuberculosis, M. avium, M. kansasii and M. intracellulare, were repeatedly tested at three sites. Of 480 determination against eight agents, 455 (94.8%), 470 (97.9%) and 455 (94.8%) of the MICs read after 5-day, 7-day and 10-day incubations fell within 3log2 dilutions, respectively. The MICs gradually elevated during the incubation, however those of 7-day incubation were highly precise and easily determined. A total of 160 clinical isolates of M. tuberculosis and 114 of nontuberculous mycobacteria were tested against eight agents. As for the primary drugs (SM, INH, RFP and EB), most isolates of M. tuberculosis were highly susceptible with MIC90, < or = micrograms/ml. Both LVFX and SPFX were also active. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of RFP, LVFX, SPFX and CAM were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative and nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.
Antitubercular Agents↗
Modulation of GABAC response by Ca2+ and other divalent cations in horizontal cells of the catfish retina.
GABAC responses were recorded in cultured cone-driven horizontal cells from the catfish retina using the patch clamp technique. At a holding potential of -49 mV, a bicuculline-resistant inward current (IGABA) was observed when 10 microM GABA was applied. The amplitude of IGABA increased as the extracellular Ca2+ ([Ca2+]o) was increased. Concentration-response curves of IGABA at 2.5 and 10 mM -Ca2+-o had similar EC50 (3.0 and 3.1 microM) and Hill coefficients (1.54 and 1. 24). However, the maximal response estimated at 10 mM [Ca2+]o was larger than the maximal response at 2.5 mM [Ca2+]o. Increasing Ca influx through voltage-gated Ca channels and the resulting rise in the intracellular Ca2+ concentration had no effects on IGABA. However, IGABA was inhibited by extracellular divalent cations, with the following order of the inhibitory potency: Zn2+ > Ni2+ > Cd2+ > Co2+. The inhibitory action of Zn2+ on the [Ca2+]o-dependent IGABA increase was noncompetitive. The action of [Ca2+]o on IGABA was mimicked by Ba2+ or Sr2+. These results demonstrate that the extracellular domain of GABAC receptors has two functionally distinct binding sites represented by Ca2+ (facilitation) and Zn2+ (inhibition). Since [Ca2+]o and [Zn2+]o change into the opposite direction by light, it seems likely that they modify cooperatively the efficacy of the positive feedback consisting of the GABAC receptor.
Animals↗
Determination of pepstatin-sensitive carboxyl proteases by using pepstatinyldansyldiaminopropane (dansyl-pepstatin) as an active site titrant.
N-Pepstatinyl-N'-dansyldiaminopropane (dansyl-pepstatin) was prepared by the coupling of pepstatin A and N-dansyl-diaminopropane. The dansyl-pepstatin obtained strongly inhibited pepsin activity by forming a 1:1 complex. The fluorescence of the dansyl group (excitation at 320 nm, and emission near 520 nm) increased with the formation of the complex. The increase in fluorescence of dansyl-pepstatin solution was proportional to the amount of added pepsin, chymosin and cathepsin D until dansyl-pepstatin was saturated by these enzymes and at higher protease concentrations the fluorescence did not increase further. Therefore, the net amounts of active pepstatin-sensitive carboxyl proteases could be determined by detecting the inflection point of increased fluorescence upon addition of the protease to a dansyl-pepstatin solution of known concentration. Moreover, the protease concentrations of many samples were obtained easily by measurements of increased fluorescence compared with that caused by authentic protease solution. The minimum detectable amount of pepsin was about 20 pmol. On the other hand, the fluorescence did not increase upon mixing with inactivated pepsin, chymotrypsin, or trypsin. The K(i) value of dansyl-pepstatin for pepsin was similar to that of pepstatin A. It was possible to determine the amount of chymosin contained in rennet by this method. The inactivation curve of pepsin in pH 6.5 buffer was also determined quickly and easily by the use of this method. This assay method for pepstatin-sensitive carboxyl proteases is very simple and easy, and it is possible to determine the net amounts of active pepstatin-sensitive carboxyl proteases even in crude mixtures.
Animals↗
Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.
In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.
Amino Acid Sequence↗
Substrate specificity of honeydew melon protease D, a plant serine endopeptidase.
The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.
Amino Acid Sequence↗
Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.
A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.
Amino Acid Sequence↗
Fusaricidins B, C and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8: isolation, structure elucidation and biological activity.
Fusaricidins B, C and D, new depsipeptide antibiotics, have been isolated as minor components from the culture broth of Bacillus polymyxa KT-8 which was obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum. The structure of fusaricidin B has been elucidated mainly by various NMR experiments coupled with amino acid analysis in relation to fusaricidin A, the main component of the complex, whose structure was reported previously. The fraction consisting of fusaricidins C and D was unsuccessfully separated, giving roughly a 4:1 mixture of the two, respectively. The structures of fusaricidins C and D have been determined within the mixture by detailed analyses of the 2D NMR spectra. Fusaricidins B, C and D are active against fungi and Gram-positive bacteria almost as well as fusaricidin A.
Anti-Bacterial Agents↗
Appearance of morphologically abnormal Sertoli cells in infertile PD male rats during postnatal development.
Our previous studies using 12-week-old PD (pd/pd) male rats have indicated high incidence of morphologically abnormal Sertoli cells. Because sterility in these males is attributable to abnormal spermatogenesis, it is suggested that the abnormal Sertoli cells are related to abnormal spermatogenesis. In this study, the testes of prepubertal 7- and 21-day-old male pd/pd rats were examined ultrastructurally for the presence of abnormal Sertoli cells. The pd/+ males served as controls. At 7 days of age, light and dark types of Sertoli cells were found in pd/pd males. However, no anatomic abnormality existed in those cells. Cytoplasmic organelles were underdeveloped. The only difference between the light and dark Sertoli cells was the high electron density in the nucleus and cytoplasm of the dark cells. These features were similar between pd/pd and pd/+ males. At 21 days of age, the dark Sertoli cells had disappeared, and light Sertoli cells and abnormal Sertoli cells were observed in pd/pd males. The light Sertoli cells had well-developed cytoplasmic organelles. On the other hand, abnormal Sertoli cells had a dark appearance, irregularly shaped nuclei, and a large quantity of lipid droplets in the cytoplasm. These findings suggested the presence of functional disorders in lipid metabolism. The light and abnormal Sertoli cells also were found in pd/+ males; however, incidence of the abnormal Sertoli cells was significantly higher in pd/pd males (11.3%), compared with that in pd/+ males (4.9%). Thus, these results indicate that in pd/pd male rats, a number of abnormal Sertoli cells appear by 21 days of age.
Animals↗
Fusaricidins B, C and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8: isolation, structure elucidation and biological activity
Fusaricidins B, C and D, new depsipeptide antibiotics, have been isolated as minor components from the culture broth of Bacillus polymyxa KT-8 which was obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum. The structure of fusaricidin B has been elucidated mainly by various NMR experiments coupled with amino acid analysis in relation to fusaricidin A, the main component of the complex, whose structure was reported previously. The fraction consisting of fusaricidins C and D was unsuccessfully separated, giving roughly a 4:1 mixture of the two, respectively. The structures of fusaricidins C and D have been determined within the mixture by detailed analyses of the 2D NMR spectra. Fusaricidins B, C and D are active against fungi and Gram-positive bacteria almost as well as fusaricidin A.
Journal Article↗
Health impact of 1996 tornado in Bangladesh.
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Aged↗
Effect of RNA editing and subunit co-assembly single-channel properties of recombinant kainate receptors.
1. Patch-clamp methods have been used to examine single-channel properties of recombinant GluR5 and GluR6 kainate-preferring glutamate receptors which differ in a single amino acid residue as a result of RNA editing at the Q/R (glutamine/arginine) site. Subunits were expressed alone or in combination with the high-affinity kainate receptor subunit KA - 2 in transfected human embryonic kidney (HEK-293) cells. 2. In outside-out patches, unedited homomeric GluR6(Q) receptors exhibited directly resolved domoate-activated single-channel conductances of 8, 15 and 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductance of 5.4 pS, while the edited isoform GluR6(R) had an unusually low channel conductance (225 fS). 3. Homomeric channels composed of GluR5(Q) subunits exhibited three conductance states of 5, 9 and 14 pS characterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reported to form functional homomeric receptors, had an extremely low conductance (< 200 fS). 4. Heteromeric GluR6(Q)/KA-2 kainate receptors gave single-channel events indistinguishible from homomeric GluR6(Q) channels. Conversely, openings produced by GluR5(Q)KA-2 and GluR5(Q) receptors differed from each other in their kinetic properties. The primary effect of co-expression of KA-2 with GluR5(Q) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single-channel conductances of heteromeric edited receptor-channels; channel conductances were 950 fS for GluR5(R)KA-2 receptors and 700 fS for GluR6(R)/KA-2 receptors. Both receptor types had significantly higher conductances than the respective homomeric channels, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing dramatically reduces single-channel conductance. Furthermore, we find similarity between the kainate receptor-channels described in sensory neurones and the recombinant GluR5(Q) homomeric channel. Characterization of recombinant single-channel properties could therefore aid identification of the native kainate receptors.
Action Potentials↗