Biomedical subjects
Publications and source records attributed to M Kuroda.
The MeOH extract of Epimedium sagittatum was found to show neurite outgrowth activity on cultured PC12h cells. Bioassay-guided fractionation of the MeOH extract yielded six prenylated flavonol glycosides, ikarisoside A (1), icarisid II (2), epimedoside A (3), icariin (4), epimedin B (5), and epimedokoreanoside-I (6) as the active ingredients.
A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA-DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4-type phages having a more elongated head.
Novel neutral glycosphingolipids isolated from the plerocercoids of a tapeworm, Spirometra erinacei, may be expected to be involved in host-parasite interactions. We have synthesized this glycosphingolipid analogue containing 2-branched fatty alkyl residue in place of ceramide. Glycosylation of nonreducing-end trisaccharide derivative 15 with the reducing-end disaccharide derivative 17 in the presence of trimethylsilyl triflate (TMSOTf) gave the desired oligosaccharide derivative in good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycoside 19 was converted to glycosylimidate 20, which was condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated to give the target glycosphingolipid analogue 22.
Using a new system developed by us for acquiring microscopic images automatically, we compared the morphological changes that apoptotic cells undergo with changes in the staining pattern of annexin V-enhanced green fluorescent protein (AV-EGFP) and propidium iodide (PI) in individual cells. Jurkat cells were treated with 5 mM CaCl2 alone, anti-Fas antibody and heating at 42 degrees C for 30 min or 46 degrees C for 60 min, and then were incubated in medium with 5 mM CaCl2. Time-lapse DNA fragmentation analysis and morphological observation revealed that the anti-Fas antibody and heating at 42 degrees C for 30 min induced typical apoptosis in the cells, and heating at 46 degrees C for 60 min induced typical necrosis. Time-lapse observation of individual cells stained with AV-EGFP and PI confirmed that apoptotic cells were stained at first with AV-EGFP alone, and thereafter also with PI when the cellular membrane ruptured and the cell underwent secondary necrosis. Most of the cells which underwent necrosis were stained simultaneously with AV-EGFP and PI. There was a significant time interval between the staining of individual cells with AV-EGFP, indicating apoptosis, and staining of these cells with PI, which indicated the occurrence of secondary necrosis. These results suggest that time-lapse examinations are necessary to distinguish apoptosis, secondary necrosis and necrosis in cells from one another. This study presents direct evidence that apoptotic cells undergo secondary necrosis, which could be recognized with PI.
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Laminar distribution and synaptic organization of non-cholinergic projections to the medial limbic cortex from the basal forebrain (BF) were studied in rats. To eliminate BF cholinergic neurons selectively, a immunotoxin, 192IgG-saporin, was injected into lateral ventricle, and 7 days later, to label the remaining non-cholinergic elements, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into BF. In rats injected with the toxin, PHA-L labeled fibers, presumably originating from the remaining non-cholinergic neurons, were distributed in layers V-VI of the medial limbic cortex. Ultrastructural observation showed that the PHA-L labeled fibers were classified into two categories. One class of the axons provided with numerous passant boutons, whereas the other carried fewer passant varicosities. Both classes of terminals were found to make symmetrical synaptic contacts predominantly with dendritic shafts, together with a large number of degenerating terminals making asymmetrical synapses with dendritic spines, presumably cholinergic in nature. These results suggest that non-cholinergic projections from the BF exert a modulatory effect directly on cortical neurons in the medial limbic cortex.
BACKGROUND: Nephron-sparing surgery for incidentally detected small renal tumors has been performed. The main objection to such surgery concerns the incidence rate of satellite renal tumors. In this study, the authors analyzed the rate of incidence and proliferative potential of satellite renal tumors. METHODS: The tumors of 124 renal cell carcinoma patients with a clinically identified unilateral and single tumor measuring </=90 mm who were examined between November 1991 and May 1997 were analyzed prospectively. The authors determined whether these specimens obtained by radical nephrectomy had satellite tumors, and whether the satellite tumors were reactive with a monoclonal antibody (MoAb), MIB-1, to assess their proliferative potential. RESULTS: Satellite renal cell carcinomas were detected in 8 of the 124 patients (6.5%). None of the pathologic variables examined (tumor grade, tumor stage, main tumor size, cell pattern, and vascular invasion) were found to be predictive of the presence of satellite tumors. Of these eight tumors, two main tumor specimens and three satellite tumor specimens reacted with the MIB-1 MoAb. The reactivity of MIB-1 correlated with the tumor grade. The satellite tumors were observed to have a proliferative potential compared with the main tumors. Among 86 kidney specimens from patients with urothelial tumors after total nephroureterectomy, 2 (2.3%) contained small renal cell carcinoma but did not show positive staining with the MoAb MIB-1. CONCLUSIONS: The incidence rate of satellite tumors among 124 renal cell carcinoma patients was 6.5% and the presence of satellite tumors was not predictable. Some of these satellite tumors showed positive staining with the MIB-1 MoAb, but small renal tumors detected in kidney specimens with urothelial tumors did not.
Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.
The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation.
BACKGROUND: In the TNM classification of renal cell carcinoma released in 1997, T1 tumors were defined as organ-confined tumors 7.0 cm or less in size, and T2 as those larger than 7.0 cm. The consideration of tumor size should be predicated on its prognostic value in predicting survival, because the goal of clinical staging is to separate patients into similar classes of survival based on the extent of disease at presentation. The authors examined the impact of tumor size on the clinical outcomes of patients with Robson Stage I disease to determine a size cutoff that would maximize the predictive value of the TNM staging system. METHODS: Between 1962 and 1995, 382 patients with renal cell carcinoma were treated at the Department of Urology at the Osaka Medical Center for Cancer and Cardiovascular Diseases in Osaka, Japan, and the TNM staging of 350 of those patients was recorded. Of 350 patients, 157 (45%) were at TNM Stage I, 47 (13%) at Stage II, 65 (19%) at Stage III, and 81 (23%) at Stage IV, according to the TNM stages defined in 1997. Robson Stage I includes TNM Stages I and II, and 204 patients in these stages were analyzed in this study. This study group included 146 men and 58 women with a mean age of 57.8 years (range, 26-84 years). The mean follow-up period was 5.4 years. RESULTS: The patient survival periods were not significantly different for those with TNM Stages I and II. This finding indicated that the tumor size cutoff of 7.0 cm was not useful in predicting the prognosis. The patients at TNM Stages I and II were then divided into two groups at each size cutoff, from 2.5 cm to 9.0 cm, at 0.5-cm intervals. The tumor size cutoff of 5.5 cm was most predictive of patient survival (P = 0.0121). None of other patient characteristics varied significantly between the two groups at this dichotomous point. Tumor size and microscopic intrarenal venous invasion, but not grade or infiltration pattern, were found in univariate and multivariate analyses to be significantly predictive of the survival of Robson Stage I patients after radical nephrectomy. CONCLUSIONS: The current data indicate that the tumor size cutoff of 5.5 cm was most significantly predictive of the survival of Robson Stage I patients after radical nephrectomy. The tumor size cutoff of 5.5 cm was also shown to be significant in univariate and multivariate analyses.
Protection of hepatocytes from ischemia-reperfusion injury is a clinically important issue. The purpose of this study was to evaluate changes in acute liver damage and recovery after ischemia-reperfusion in rats with asialoglycoprotein receptor (ASGP-R) ligand. Ischemia was induced by clamping the hepatoduodenal ligament for 90 min. At 1, 3, 24, 48 hr, 1 and 2 wk after reperfusion, I-125-GSA was injected. Five min after injection, blood samples were obtained and the liver was removed. Several regions from each lobe were dissected, weighed and counted. Mean uptakes (% dose/g) in the liver and blood samples were calculated. Histologic sections stained with hematoxylin-eosin (H-E) stain showed ischemic damage at 1 and 3 hr, and focal hepatocyte necrosis at 24 hr. Predominant massive necrosis was not seen. The mitotic index with H-E stain and proliferating cell nuclear antigen (PCNA) labeling index were highest at 1 wk, indicating liver regeneration. At 1 and 3 hr, liver uptake was significantly decreased, and blood uptake was significantly increased, indicating decreased tissue blood flow and ischemic damage. Liver uptake showed significant increases at 48 hr and 1 wk, and was the highest at 1 wk, indicating liver regeneration during the convalescence stage. ASGP-R binding may provide valuable information on ischemia-reperfusion injury and recovery.
The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi.
N-terminal amino acid sequences of major capsid proteins (Mcps) of three vibriophages (KVP20, KVP40 and nt-1), two aeromonad phages (Aeh 1 and 65) and coliphage T4 were compared. All these phages are morphologically similar, belonging to family Myoviridae and the vernacular genus name "T4-like phages". A dendrogram constructed from homology data indicated that (i) the three vibriophages were closely related, (ii) the two aeromonad phages were also fairly related and (iii) these five phages were all distantly, but definitely, related to coliphage T4. These results suggest that Mcps of morphologically similar phages are highly conserved and may serve as a measure to assess the phylogenetic relationships among different phages of similar morphology.
Vibriophage KVP40, a large tailed DNA phage morphologically similar to T-even coliphages, has a major capsid protein (Mcp) homologous to the equivalent protein, gp23(*), of coliphage T4. The sequence analysis was extended to a 7-kbp region immediately upstream of the mcp gene encoding the precursor of Mcp. The region as a whole was fairly homologous to the corresponding region of the T4 genome and contained 8 ORFs homologous to T4 genes 17, 18, 19, 20, 67, 68, 21, and 22 in the same order as in T4. These findings thus strongly suggest that these two phages are phylogenetically related.
Two isoforms of the human cadherin-11/OB-cadherin gene, the intact and the variant forms, had been isolated from an osteosarcoma cDNA library. The intact form has a typical cadherin structure, whereas the variant form, generated by alternative splicing, encodes a cytoplasmic domain that is completely different from that of the intact form and lacks a homophilic cell-cell adhesion ability. At the protein level, the secreted form generated from the intact cadherin-11 is present. We examined the expression of the intact and the variant forms of cadherin-11 in 23 primary and metastatic osteosarcomas from 22 patients by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, revealing that all 23 tumors in the patients expressed the variant form and three of them expressed it prominently. On the other hand, Western blot analyses of six tumors showed that the secreted form was strongly expressed, and furthermore, expression of N-cadherin was extremely low. Overexpression of the intact cadherin-11 cDNA in osteosarcoma cell lines demonstrated that the secreted form is derived from the intact form of cadherin-11 in osteosarcoma. Immunohistochemically, cadherin-11, N-cadherin, and beta-catenin were expressed at the cell surface of fetal osteoblasts, whereas in osteosarcoma cells, they were expressed only focally or weakly in the cytoplasm. Considering the function of cadherin in carcinomas, it is suggested that the anomalous expression of human cadherin-11 in osteosarcoma and the reduced expression of N-cadherin play a role in metastasis and the irregular morphology in the highly malignant mesenchymal tumor.
Chemical examination of the aerial parts of Dracaena draco has led to the isolation of a total of nine steroidal saponins, including five new ones. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L -arabinopyranosyl} 24-O-beta-D-fucopyranoside, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta, 23,24-tetrol 1-O-{O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L -arabinopyranoside}, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(4-O- acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyransoide} , (23S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-alpha-L- rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyranoside} and (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-(4-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L- arabinopyranoside}. The isolated saponins were evaluated for their cytostatic activity on leukemia HL-60 cells.
Five new spirostanol saponins and a new furostanol saponin were isolated from the fresh bulbs of Lilium candidum. Their structures were elucidated on the basis of spectroscopic analysis, including two-dimensional NMR spectroscopic techniques and the result of acid hydrolysis. The isolated saponins contained a branched triglycoside moiety assigned as O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->6)]-beta - D-glucopyranose with the formation of an O-glycosidic linkage to C-3 of the aglycone as the common structural feature. The inhibitory activity of the saponins on Na+/K+ ATPase was evaluated.
A new spirostanol saponin was isolated from the underground parts of Ruscus aculeatus and the structure was assigned as (23S,25R)-spirost-5-ene-3 beta,23-diol 23-O-[O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside] on the basis of spectroscopic analysis, including two-dimensional NMR spectroscopic techniques and the result of acid hydrolysis. The saponin is unique in structure having a diglycoside unit at C-23 of the spirostanol skeleton.