Biomedical subjects
Publications and source records attributed to K Nishimura.
A toxic substance was purified from larvae of the antilion, Myrmeleon bore, by DEAE Sephacel and Phenyl Superose column chromatography. The substance was a large polypeptide with a molecular weight of about 165-167 kDa. Its paralytic activity measured by injection against German cockroaches was about 130 times higher than that of tetrodotoxin on a molar basis.
Eight muteins of recombinant human tumor necrosis factor-alpha (rhTNF; 1SSSRTP...29RR...155L), in which 29Arg was replaced by another amino acid, were prepared and their anti-tumor effects in BALB/c mice bearing Meth A fibrosarcoma were evaluated. The therapeutic indices, which mark the extent of the therapeutically effective dose, of V29 (29Arg-->Val) and D29 (-->Asp) were 3.5 and 3.2, respectively, whereas that of rhTNF was 1.4. Clearly, the therapeutically effective range of these muteins was extended along with a decrease in lethal toxicity. V29 did not produce hypotension in the rat system, but D29 did. In addition, V29 showed potent anti-tumor activity (Tumor Volume Inhibition Rate = 81% on day 15 after implantation) in 3 consecutive injection schedules despite the decreases in toxicity compared with rhTNF. The relative receptor binding constant was determined using HEp-2 cells (expressing mainly 55-kDa-TNF receptor; p55R) and HL60 cells (expressing mainly 75-kDa-TNF receptor; p75R), and revealed that the reduced toxicity of V29 in mice was due to the reduced binding to p55R (34% of rhTNF). On the other hand, the ratio of the constants HEp-2/HL60 of V29 was 11 in comparison with the value of 1.0 for rhTNF, suggesting that this mutein binds preferentially to p55R. The biological activities in human cell lines (HEp-2 and HL60 cells) correlated well with the binding activities to each receptor in vitro. Therefore, the much lower toxicity and the potent anti-tumor activity of this mutein suggest that V29 merits further investigation in pre-clinical and clinical trials.
Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G1 DNA, whereas large unbudded cells had either a G2 or G1 DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G1 or G2 period.
To examine the induction of terminal enzymes of the heme-biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned. The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria. Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former. The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively. When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau. mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h. The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8-fold within 12 h and remained unchanged thereafter. Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities. The magnitude of PPO induction was less than that of CPO and FEC. Thus, induction of three terminal enzymes of the heme-biosynthetic pathway is an early event in MEL cell differentiation. The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.
Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway. Hemes are essential components of redox enzymes, such as cytochromes. Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows poorly even in rich medium. By complementation with a human placental cDNA library, we were able to isolate a clone that enhanced the poor growth of such a hemG mutant strain. The clone encoded the gene for human PPO. Sequence analysis revealed that PPO consists of 477 amino acids with a calculated molecular mass of 50.8 kilodaltons. The deduced protein exhibited a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis. The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins. Northern blot analysis revealed the synthesis of a 1.8-kilobase pair mRNA for PPO. A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Furthermore, the cDNA was expressed in vitro as 51-kilodalton protein, and after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.
BACKGROUND: Accurate noninvasive determination of myocardial viability is of paramount importance for the clinical identification of patients who will benefit most from revascularization. The preserved metabolic activity in the myocardium, as studied with positron emission tomography (PET), has been considered a gold standard for this purpose. However, recent reports show that moderate hypoperfusion or stress-induced ischemia may represent reversible ischemia. The present study was undertaken to compare the value of perfusion and metabolic studies with PET for predicting improvement in wall motion after revascularization. METHODS AND RESULTS: Of 61 patients who had regional asynergy and underwent PET before revascularization, 43 patients who had successful revascularization were included in the study. Each patient underwent rest-stress 13N-ammonia perfusion scans and 18F-fluorodeoxyglucose (FDG) scan at rest while in a fasting state. Reversible ischemia was considered to be present when the resting perfusion was > or = 50% of the peak value, stress-induced hypoperfusion was present, or an increase in FDG uptake was observed. Of 130 asynergy segments, 51 segments had improved wall motion after revascularization. The positive and negative predictive values for improvement in asynergy were 48% and 87% by the rest perfusion study, 63% (P = .05 versus the rest value) and 87% by the rest-stress perfusion study, and 76% (P < .01 versus the rest value) and 92% by the FDG study. CONCLUSIONS: FDG PET provided the best predictive value for improvement in wall motion after revascularization. On the other hand, 13N-ammonia PET is useful for predicting nonreversible myocardial scarring when it shows severe hypoperfusion at rest or hypoperfusion without stress-induced ischemia.
The effects of the selective prostaglandin D2 (DP) receptor agonists, 572C85 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethyl- amino)hexahydrocyclopenta(d)imidazol-2(1H)-one) and 192C86 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethylidene - amino)-3-ethylhexahydrocyclopenta(d)imidazol-2(1H)-one), were determined on intraocular pressure regulation in rabbits and cats. 572C85 (50 micrograms) in rabbits maximally lowered intraocular pressure by 4.3 mm Hg, and significantly for 4 h compared to control. In cats 572C85 had a similar effect. 192C86 (50 micrograms) reduced intraocular pressure by 2.8 mm Hg for 2 h in rabbits. Following exposure to the specific DP receptor antagonist, BW A868C ((+/-)-3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamin o)- hydantoin; 50 micrograms), which had no effect on intraocular pressure by itself, 572C85 (50 micrograms) did not reduce intraocular pressure in rabbits and cats. The intraocular pressure lowering effect of the mixed DP and EP receptor agonist, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin; 50 micrograms), in cats was suppressed by only 64% by BW A868C (50 micrograms). These results clearly show that the DP receptors in rabbit and cat eyes are involved in intraocular pressure regulation. However, under baseline conditions DP receptor activity does not contribute to this regulation.
We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell. Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA. This new gene, designated hemK, was located at 27 min on the linkage map of the E. coli chromosome. By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases. The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA. An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities. H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.
This study was designed to evaluate the ability of cementum-impregnated gelatine membranes (CGM) to stimulate regeneration of periodontal ligament following surgery. Three monkeys with no periodontal disease were used. Following flap elevation, recession type defects were created on the buccal side of maxillary lateral incisors and second premolars, and roots were subsequently planed. Experimental sites received CGM at approximately 2-3 mm below the cementoenamel junction while control sites received gelatine membrane (GM) free of cementum. Wounds were allowed to heal for 3 weeks, during which time daily plaque control measures were maintained. Tissues prepared from sacrificed animals revealed that experimental specimens exhibited formation of new cementum, new bone, and periodontal ligament. Control specimens exhibited connective tissue adhesion without either new cementum or significant new bone formation. Histometric analysis showed that the average vertical growth of cementum (NC) and bone (NB) at experimental sites were 3.48 +/- 0.29 and 0.64 +/- 0.43 mm, respectively. At the control sites the corresponding results were NC = 0.09 +/- 0.03 and NB = 0.06 +/- 0.28. It was concluded that placement of CGM resulted in significantly greater postsurgical cementum and bone formation than that of GM alone.
Freeze-fracturing of outer wall layers of Cladosporium conidia revealed two types of ultrastructure, coinciding with taxonomic characteristics. The outer conidial layers were essentially smooth in the human pathogenic species, C. bantianum, C. carrionii, and C. trichoides. In contrast, mosaic arrays of rodlets on conidia were observed with freeze-fracturing in the saprobic species, C. cladosporioides, C. coralloides, C. herbarum, C. sphaerospermum, and C. variabile. Conidia of C. elatum were an exception among the saprobic species as they had smooth surfaces. The present study supports the suggestion that the human pathogenic Cladosporium species should be transferred to another genus.
Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 36 isolates of Exophiala spinifera (8 isolates from Brazil, 9 from China, 15 from Columbia, 1 from the United States and 2 from Venezuela). E. spinifera isolates displayed a high degree of mtDNA diversity in RFLP patterns and were clustered into six genetically heterogeneous groups (Group 1 through Group 6). Isolates of Group 2 including the type strain seemed to have a worldwide distribution. RFLP patterns of E. spinifera were also distinct from those of other dematiaceous fungi, such as E. jeanselmei, E. moniliae, E. dermatitidis, Fonsecaea pedrosoi, Cladosporium carrionii and Phialophora verrucosa. These results indicate that E. spinifera may be a taxonomic complex and that RFLP patterns will be useful in the identification, typing and epidemiology of the E. spinifera variants.
Ecdysone analogs with various side chains at the 17-position of the steroid structure enhanced the incorporation of N-acetylglucosamine as 20-hydroxyecdysone into the cultured integument prepared from Chilo suppressalis. Their activity in terms of the concentration required to give 50% of the maximum response varied with the structure. Piperonyl butoxide, an inhibitor of oxidation metabolism, did not enhance the in vitro effect of the compounds. The order of potency was ponasterone A > 20-hydroxyecdysone > cyasterone > inokosterone > makisterone A >> ecdysone.
Pre-mated adult female newts and embryos have been flown on the International Microgravity Laboratory-2 (IML-2) Space Shuttle flight in 1994 (Wiederhold et al., 1992b). With the specimens available from this flight, the calcification of otoliths, ulna, radius and backbone of the flown larvae and adult newts were analyzed. The experiments presented here studied the development of the otoliths on the ground. Otoliths of living newts, from embryo to adult, were observed in situ with the application of a new X-ray and bio-imaging analyzer system. For the establishment of this method, newts at different developmental stages were used. An imaging plate temporarily stores the X-ray energy pattern at the bio-imaging analyzer. A latent image on the imaging plate was transformed into a digital time series signal with an image reader. Acquired digital information was computed with the image processor. The processed information was recorded on film with an image recorder, in order to visualize it on an enlargement computed radiograph. To analyze development of the otoliths, photo-stimulated luminescence level was detected by an image analyzer, using transmitted X-ray photons. A single clump of otoconia could first be seen at stage 33. Stage-36 embryos first have distinguishable otoliths, with the utricle in front and saccule behind. Our results show that this X-ray method detects the otoliths equally as well as sectioning. In the newt, the mandibular/maxillary bone formed before the spine. It is suspected that for the newt embryo, living in water, feeding becomes necessary prior to support of the body.
In CT the presence of mediastinal or hilar lymphadenopathies and thickened bronchovascular bundles are landmarks for the diagnosis of pulmonary sarcoidosis. The major CT findings for parenchymal involvement are thickened bronchovascular bundles, large parenchymal nodules, pleural or subpleural nodules, ground-glass opacities, local lung volume loss (distortion of the lung parenchyma), and microscopic and macroscopic honeycombing. The thickened bronchovascular bundles correspond histologically to granulomas, either with or without perigranulomatous fibrosis in the connective tissue sheath around the pulmonary vessels and airways. Conglomerate granulomas are represented on CT by high-attenuation nodules, and the ground-glass opacities are caused by the summation of a number of small granulomas in the interstitium.
Right ventricular function during left heart bypass was evaluated by volumetric analysis with a conductance catheter in 12 dogs with postischemic cardiac dysfunction. The conductance catheter was used to assess the volumetric status of the right ventricle and thereby allowed a right ventricular pressure-volume curve to be obtained, in which transient volume loading on the right ventricle was applied. The following right ventricular properties during left heart bypass were assessed and compared with properties measured without left heart bypass, by means of load-independent parameters: maximum elastance, stroke work/end-diastolic volume relation, end-diastolic pressure/volume relation, and stroke work/end-diastolic pressure relation. The stroke volume derived from the conductance catheter and the electromagnetic flow probe showed good linear correlation (r2 = 0.733 to 0.975). After initiation of left heart bypass, maximum elastance did not change significantly, although volume intercept significantly increased, from 1.2 +/- 7.3 to 3.6 +/- 7.9 ml (p < 0.05). End-diastolic pressure/volume relation was well fitted to the exponential curve (EDP = e(k1.EDV+k2)) and was shifted to the right and downward during left heart bypass; the slope k1 significantly decreased, from 0.12 +/- 0.06 to 0.10 +/- 0.07 (p < 0.01). Stroke work/end-diastolic volume relation and stroke work/end-diastolic pressure relation were closely fitted to the linear regression, and their slopes were significantly increased during left heart bypass, from 0.14 +/- 0.08 to 0.18 +/- 0.08 (p < 0.05) and from 0.22 +/- 0.15 to 0.34 +/- 0.19 (p < 0.01), respectively. These results suggest that the decompression of the left ventricle and septal shifting by left heart bypass provide good diastolic compliance and good systolic performance because of afterload unloading of the right ventricle. Thus the left heart bypass improved the overall right ventricular performance, particularly at higher end-diastolic pressures, in dogs with postischemic cardiac dysfunction.
SETTING: KL-6, a human MUC-1 mucin preferentially expressed on type II pneumocytes, is a sensitive serum marker for evaluating alveolar damage of interstitial pneumonia and pulmonary fibrosis. Some patients with pulmonary tuberculosis develop severe respiratory dysfunction caused by extensive pulmonary fibrosis, compensatory emphysema and fibrous pleural thickening. OBJECTIVE: To evaluate the clinico-pathological significance of KL-6 in pulmonary tuberculosis. DESIGN: Serum KL-6 levels were measured in sera from 57 patients with active pulmonary tuberculosis and 38 healthy controls by a sandwich-type enzyme-linked immunosorbent assay. Immunohistochemistry was performed by an avidin-biotin-peroxidase complex method. RESULTS: KL-6 levels were significantly higher in the patients than in the healthy controls (518 +/- 693 [SD] vs 227 +/- 91 U/ml, P < 0.001) and increased significantly according to the extent of pulmonary lesions evaluated by chest X-ray (P < 0.001). There was a significant negative correlation between serum KL-6 levels and % vital capacity (VC) (r = 0.642, P < 0.05). KL-6 was strongly expressed on proliferated type II pneumocytes and cuboidal epithelial cells adjacent to thickened intralobular septa and pleura. CONCLUSIONS: In pulmonary tuberculosis, serum KL-6 originates from proliferated type II pneumocytes and cuboidal epithelial cells, and is a useful marker presenting the degree and extent of pulmonary fibroproductive lesions.
RATIONALE AND OBJECTIVES: For intravenous (i.v.) coronary arteriography, a real-time two-dimensional (2D) imaging system is being developed using above-K-edge monochromatic X-rays alone. The potential diagnostic value of this system was examined in the current study. METHODS: The angiographic system consisted of an asymmetric silicon (311) monocrystal, an image intensifier, and a charged-coupled device camera. It was constructed at the beam line of the Tristan Accumulation Ring. Monochromatic X-rays of 33.32 keV were used, and each image was recorded over 2 msec. RESULTS: Ventricular wall motion and the anatomy of the coronary arteries could be seen in dogs by sequential images obtained without subtraction. The left anterior descending coronary artery, left circumflex coronary artery, and right coronary artery and the branches of these vessels were observed. The parts of the coronary arteries overlapping the aorta and left ventricle were revealed somewhat during washout of the contrast material. CONCLUSION: For a 2D imaging system, monochromatic i.v. coronary angiography using an energy above the iodine K-edge might be able to image the coronary arteries without subtraction. However, the image quality needs to be improved by increasing the X-ray flux, decreasing background radiation scatter from the object, and decreasing contamination with 99-keV X-rays.
Recently we performed multi-modal therapy including chemotherapy with bone marrow transplantation (BMT), surgery, and local irradiation in the treatment of advanced neuroblastoma. As a result, all Stage III patients survived, and 4 out of 8 Stage IV patients who underwent our original systemic gross complete resection achieved long-term survival, 4y8m, 5y8m, 5y6m and 5y5m, respectively. Long-term survival patients were treated by local irradiation, brain surgery and intensive induction chemotherapy before primary site operation, and when resolution of distant metastases was obtained, the primary site was resected completely, followed by postoperative supralethal chemotherapy with BMT. The purpose of this report is to elucidate the role of surgery in advanced neuroblastoma based on our clinical results.