Biomedical subjects
Publications and source records attributed to M Kaneda.
Cucumisin (EC 3.4.21.25) isolated from prince melon fruit is a plant serine protease. Its milk-clotting activity was compared with plant cysteine proteases such as papain (EC 3.4.22.2) and ficain (EC 3.4.22.3). Cucumisin was more stable than papain under the condition of pH 7.1, 37 degrees C for 24 h. The milk-clotting activity of cucumisin was the same to that of papain and was half value of that of ficain.
CONCLUSION: Pancreatic metastasis from renal cell carcinoma is extremely rare. The average time between nephrectomy and the diagnosis of metachronous metastases is reported to exceed 10 yr. Therefore, the initial diagnosis may be neglected in the cases of prolonged disease-free interval. When it does occur simultaneously or metachronously, aggressive surgical resection, when possible, seems to be the most effective treatment for this metastatic lesion. BACKGROUND: An 81-yr-old female patient, who 8 yr previously had undergone right radical nephrectomy for renal cell carcinoma, presented with solitary pancreatic metastasis, which was successfully treated with a distal pancreatectomy. Only 66 cases of clinically diagnosed renal cell carcinoma metastatic to the pancreas are reported in the world literature and 49 of the patients (including ours) underwent a definitive surgical resection. Our case, treated by distal pancreatectomy, and a review of the relevant literature including all reported cases of renal cell carcinoma metastatic to the pancreas, are presented. RESULTS: The patient was well without any evidence of recurrence at 22 mo after the operation.
Fusaricidin A, a new depsipeptide antibiotic, was isolated from the culture broth of Bacillus polymyxa KT-8 obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum. The structure of fusaricidin A was determined by 1D and 2D NMR and MS experiments coupled with amino acid analysis to be a hexadepsipeptide containing 15-guanidino-3-hydroxypentadecanoic acid as a side chain. The absolute configuration of each amino acid residue was determined by chiral HPLC. Fusaricidin A is active against fungi and Gram-positive bacteria.
Recently, high dose rate endobronchial brachytherapy has been carried out for the treatment of lung cancer. We devised a new applicator for Ir-192 high dose rate brachytherapy that can position the source in the center of the bronchial lumen, and tried to set up reference points according to bronchial diameter, for optimal dose distribution. Treatment consisted of external beam radiotherapy (40-60 Gy in 4-6 weeks) and endobronchial brachytherapy (6 Gy x 3 fractions for curative intent, 10 Gy x 1 fraction for palliative intent). Reference dose points were 3, 5, 7 and 10 mm from the center of the source according to bronchial diameter. We treated 19 patients with endobronchial brachytherapy and used the new applicator in 13 of them. We could place the applicator in all 13 patients, and no remarkable side effects were observed during the observation period. Our newly designed applicator will help to reduce radiation side effects caused by irradiation overdose due to adherence of the source to the bronchial wall. The reference point should be set according to bronchial diameter not only for curative intent but also palliative intent.
We obtained a successful response to PUVA therapy in a 51-year-old Japanese man who had multiple nodules of the trunk, extremities and scalp along with polyclonal hypergammaglobulinaemia; the scalp lesions were associated with extensive alopecia. Examination of skin biopsies showed a dense infiltrate of mature plasma cells in the dermis and hair follicles; no systemic disease or functional involvement of other organs was detected. The clinical and histological findings were compatible with cutaneous plasmacytosis and treatment with topical PUVA gradually reduced the size and number of the lesions.
A monoclonal anti-idiotypic antibody, Id8F7, previously shown to bind to a phosphatidylserine (PS)-specific binding site on protein kinase C (PKC) has been used to identify a 12-amino acid consensus sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The 14-amino acid synthetic peptide derived from the corresponding region of PSD (amino acids 351-364 of the enzyme from Chinese hamster ovary cells) bound effectively and specifically to PS, and that derived from rat PKC gamma (amino acids 227-240) bound weakly but specifically to PS. Analysis of binding of Id8F7 to various synthetic peptides revealed that the consensus sequence motif, FXFXLKXXXKXR, is responsible for the interaction with both Id8F7 and PS. The results suggest that the conserved amino acid residues represent a basic structural motif for the specific interaction with PS, and the corresponding regions of PKC and PSD form the PS-specific binding sites of these enzymes.
1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR-95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR-95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS)
Heritable oligodactylism in male rats was discovered in Wistar-derived rats. In this study, external and skeletal features of the limb anomalies were examined to clarify the type of oligodactyly. External observation revealed absence and hypoplasia (with respective percentage incidence) in digits II (100, 0), III (87, 13), and IV (23, 1) of the forefeet and in digits II (26, 34), III (34, 11), and V (0, 3) of the hindfeet. Fusion was also observed in digits IV and V in the forefeet (4%) and in digits I and II in the hindfeet (15%). Skeletal examination disclosed absence of all three (proximal, middle, and distal) phalanges in digits II (97%), III (93%), and IV (67%) and of the middle phalanx in digit V (12%) of the forefeet. In the hindfeet, the missing pattern of phalanges in digits II and III was similar to, but less severe than that in the forefeet; the incidence of absence of the three phalanges in digits II and III was 43% and 45%, respectively. Digit V was more affected than that in the forefeet; absence of the middle phalanx was found at 30%. Although no absence was found in the bones proximal to the phalanges, hypoplasia and/or fusion were detected with various degree and incidences among carpi/metacarpi and tarsi/metatarsi. Based on these observations, the types of limb anomalies in this mutant rat were classified as oligodactyly of digits II-V in the forefeet and II, III, and V in the hindfeet with hypoplasia and/or fusion of the carpi/metacarpi and tarsi/metatarsi.
We present herein the case of a 66-year-old woman with disseminated malignant melanoma in whom a metastasis in the ileal mesentery ruptured into the peritoneal cavity causing an acute abdomen. The patient suddenly developed lower abdominal pain and a computed tomography (CT) scan of the pelvis confirmed the presence of an intrapelvic abscess. At emergency laparotomy, a 10 x 10 cm ruptured metastatic melanoma was found in the ileal mesentery, which demonstrated no communication with the ileum itself. To our knowledge, no other case of an acute abdomen being caused by the rupture of mesenteric metastatic melanoma lacking any communication with the bowel lumen has ever been reported.
Nine sites of oxidized insulin B-chain were cleaved by phytolacain R, isolated from pokeweed, after 20 hr of hydrolysis. Five cleavage sites resembled those of papain. Substrate specificity of phytolacain R was similar to that of papain, preferring hydrophobic P2 residues. The activities of fibrin formation and elastin hydrolysis of phytolacain R were higher than those of papain. The amino terminal sequence of phytolacain R, although similar was not identical with that of papain.
We have established a series of monoclonal antibodies that bind to phosphatidylserine (PS). One mAb, PS4A7, showed a strict specificity for PS and distinguished the stereospecific configuration of its serine moiety. We determined the amino acid sequences of the heavy and light chain variable regions of PS4A7, and examined the reactivity of the synthetic peptides corresponding to the complementarity determining region (CDR) of the mAb with phospholipids. We found that a 12-amino acid synthetic peptide corresponding to the third CDR of the heavy chain (amino acid residues 93-102, referred to as CDR3-H) bound specifically to PS. Although the affinity of the peptide to PS was markedly lower, the peptide was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS), but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine, showing a similar specificity to that of PS4A7. The specific binding of the CDR3-H peptide to PS was confirmed by ELISA and TLC-immunostaining assay. The interaction between the CDR3-H peptide and water-soluble PS-derivatives was investigated by inhibition of the ELISA. PS effectively inhibited the binding and phosphoserine showed a weak but significant inhibition, but no appreciable inhibition was observed with serine. These observations suggest that the CDR3-H peptide plays a major role in the interaction of PS4A7 with the phosphoserine residue of the PS molecule.
A radioactive peptidyl chloromethyl ketone derived from substrates of cucumisin, 3H-labeled Z-Ala-Ala-Pro-Phe-chloromethyl ketone (3H-ZAAPFCK), was synthesized. When cucumisin was incubated with a 100-fold molar excess of 3H-ZAAPFCK for 16 h, 98% of the cucumisin activity was inhibited and about 0.93 mol of 3H-ZAAPFCK was incorporated in 1 mol of cucumisin. The 3H-ZAAPFCK-modified cucumisin was reduced and pyridylethylated, and then digested by trypsin. The radioactive peptide fragment was isolated and its amino acid sequence was determined. The radioactive fragment contained 32 amino acid residues and the sequence around the labeled residue was found to be -Asp-Thr-Asn-Gly-(His)-Gly-Thr-His-Thr-Ala-. This sequence is analogous to that around the reactive site histidine residue of the subtilisin family.
Cucumisin was isolated from prince melon sarcocarp by means of a simple purification procedure. Serine protease inhibitors such as soybean trypsin inhibitor, ovomucoid, and aprotinin had no effect on the enzyme activity. alpha 2-Macroglobulin showed 38% inhibition of the original caseinolytic activity of cucumisin. The favorable synthetic substrates for cucumisin were Glt-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA. The constant (kcat/Km) for Suc-Ala-Pro-Ala-pNA was found to be 30 times greater than that for Suc-Ala-Ala-Ala-pNA. The substrate specificity of cucumisin for oligopeptides and proteins was shown to be broad.
Ganglion cells, retrogradely labeled with DiI, were dissociated from the cat retina. Under the whole-cell clamp mode using a patch pipette, ACh induced a transient current (IACh) in all cells tested. The soma diameter of ACh-responsive cells distributed from 11 to 23 microns, indicating that the preparation included Y, X, and W cells. IACh was inward rectifying. The reversal potential of IACh was close to the zero-current potential predicted from the intra- and extracellular monovalent cation concentrations by the Goldman-Hodgkin-Katz equation. The concentration-response curve gave a Hill coefficient of 0.89 and an EC50 of 3.2 x 10(-5) M. Nicotine (10(-4) M) induced a transient current, while muscarine (10(-4) M) was ineffective. The nicotinic antagonist, hexamethonium, reduced the amplitude of IACh in a concentration-dependent manner with an IC50 of 1.3 x 10(-6) M. The muscarinic antagonist, atropine, showed a concentration-dependent reduction in the amplitude of IACh with an IC50 of 4.4 x 10(-6) M. The amplitude of IACh was dependent on the extracellular Ca2+ concentration ([Ca2+]O); small at low [Ca2+]O and large at high [CA2+]O. The time course of desensitization was also dependent on [Ca2+]O; fast at low [Ca2+]O and slow at high [Ca2+]O. The replacement of extracellular Ca2+ to Sr2+ increased the amplitude of IACh, but replacement to other divalent cations reduced it. The results indicate the presence of functional neuronal nicotinic ACh receptors in Y, X, and W cells.
Bacilopeptins, new iturin-group antifungal antibiotics, were isolated from the culture broth of Bacillus subtilis FR-2 obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum. Their structures were elucidated to be cyclic lipopeptides similar to bacillomycin L by NMR and mass spectral studies coupled with amino acid analysis. The absolute configuration of each amino acid residue was determined by chiral HPLC.
Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. 'Prince Melon'). The enzyme is stable over a wide pH range (4-11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha-chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z-Tyr-ONp) and benzoyltyrosine ethyl ester (Bz-Tyr-OEt) were cleaved, but Z-Lys-ONp and tosylarginine methyl ester (Tos-Arg-OMe) were not cleaved by cucumisin. Oxidized insulin B-chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C-termini of all the valine residues in the oxidized insulin B-chain molecule.
The NC-eob mice are mutants having open eyelids at birth with complete penetrance. Detailed scanning electron microscopy and light microscopy for study of the eyelid development of NC-eob embryos/fetuses were performed at days 13 to 17 of gestation and compared with normal NC mice, with the aim of investigating the etiopathogenesis of the open eyelids at birth. No difference was observed between NC and NC-eob embryos by scanning electron microscopy and histologic examination at days 13 and 14 of gestation. At day 15 the epithelium at the tip of the eyelids had 2 to 3 layers, and clumps of round periderm cells appeared on the outer surface of the margin of the lids in NC and NC-eob embryos. However, these clumps in NC-eob embryos were not as remarkable in number and in size compared with NC embryos. At day 16 the eyelids of NC fetuses extended over the cornea and began to fuse. The epithelium at the tip of the eyelids consisted of 4 to 5 layers with the following elongated area. The surface of the tip of the elongated epithelium was covered with a large number of round periderm cells. In NC-eob fetuses the epithelium remained stationary at 2 to 3 layers. Only a small number of round cells were noted at the tip of the eyelids, and fusion did not occur. At day 17, although the upper and lower eyelids of NC fetuses were entirely fused, no change occurred in NC-eob fetuses, either in the periderm or in the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)
We classified the genotype of neutrophil Fc gamma receptor III (FcRIII) (CD16) with a new method. Genomic DNA from mononuclear cells of 39 unrelated healthy donors (13 NA1/NA1, 13 NA2/NA2, and 13 NA1/NA2 typed serologically) were subjected to polymerase chain reaction (PCR) to amplify the polymorphic third exon of the FcRIII genes. The PCR products were heat denatured, electrophoresed, and visualized by silver staining. Allelic differences were detected by distinctive electrophoretic patterns of each single strand, depending on their sequence specific conformations (single-strand conformation polymorphism [SSCP]). The genotypes of neutrophil FcRIII determined by this method were consistent with the phenotypes of NA antigens determined by serologic examinations. These results indicate that the PCR-SSCP system is a very useful tool for genotyping of the neutrophil FcRIII.