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Biomedical subjects

K Fujimoto

Publications and source records attributed to K Fujimoto.

At least 307 records · Page 17Linked to original sources
PubMed1997

Telomerase activity in human leukemic cell lines is inhibited by antisense pentadecadeoxynucleotides targeted against c-myc mRNA.

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats to the ends of chromosomes during the replicative phase of the cell cycle. We tested the hypothesis that proto-oncogene c-myc plays an essential role in the regulation of telomerase activity in vertebrate cells by exposing three human leukemic cell lines, HL60, U937, and K562, to 15-mer antisense c-myc oligonucleotides. All the treated cells showed a profound decrease in telomerase activity after c-myc antisense oligomer treatment, whereas cells treated with c-myc sense oligomers showed essentially no change in telomerase activity.

Cell Line, Transformed↗
PubMed1997

Probing of the membrane topology of sarcoplasmic reticulum Ca2+-ATPase with sequence-specific antibodies. Evidence for plasticity of the c-terminal domain.

The topology of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles was investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membranous portions of the protein. The antisera in competitive enzyme-linked immunosorbent assays only reacted with intact SR vesicles to a limited extent, but most epitopic regions were exposed by low concentrations of nondenaturing detergent, octaethylene glycol dodecyl ether (C12E8) or after removal of cytosolic regions by proteinase K. In particular, these treatments exposed the loop regions in the C-terminal domain, including L7-8, the loop region located between transmembrane segments M7 and M8, with a putative intravesicular position, which had immunochemical properties very similar to those of the C terminus with a documented cytosolic exposure. In contrast to this, the reactivity of the N-terminal intravesicular loop regions L1-2 and L3-4 was only increased by C12E8 treatment but not by proteinase K proteolysis. Complexation of Ca2+-ATPase with beta,gamma-CrATP stabilized the C-terminal domain of Ca2+-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6-7 and L7-8 loops. Immunoelectron microscopic analyses of vesicles following negative staining, thin sectioning, and the SDS-digested freeze-fracture labeling method suggested that the L7-8 epitope, in contrast to L6-7 and the C terminus, can be exposed on either the intravesicular or cytosolic side of the membrane. A preponderant intravesicular location of L7-8 in intact vesicles is suggested by the susceptibility of this region to proteolytic cleavage after disruption of the vesicular barrier with C12E8 and in symmetrically reconstituted Ca2+-ATPase proteoliposomes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca2+-ATPase domain, which under some conditions permits sliding of M8 through the membrane with cytosolic exposure of L7-8, of possible functional significance in connection with Ca2+ translocation. On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments like EGTA at alkaline pH to open up vesicles for probing of intravesicular location with antibodies.

Animals↗
PubMed1997

Eosinophil cationic protein levels in induced sputum correlate with the severity of bronchial asthma.

Activated eosinophils play an important role in the pathogenesis of bronchial asthma. In this study, we analyzed the inflammatory leukocyte population and the concentrations of eosinophil cationic protein (ECP) and albumin in induced sputum from patients with mild to severe asthma (n=36), and assessed the findings in relation to the severity of their asthma. Both the eosinophil numbers and the concentrations of ECP in the induced sputum were significantly increased in the patients with asthma compared with those in healthy subjects (n=9). There were significant positive correlations between the ECP levels and both the eosinophil counts (r=0.45) and the albumin concentrations (r=0.53). When the asthmatics were classified as having mild (n=12), moderate (n=14), or severe (n=10) asthma as evaluated by their symptoms and peak expiratory flow rate (PEFR), the ECP levels showed significant increases in accordance with the severity of asthma. The eosinophil counts in the patients with severe asthma were significantly higher than those in the patients with mild and moderate asthma; there was no significant difference between those with mild and moderate asthma. The eosinophil counts and ECP levels were also significantly positively correlated with the mean weekly total symptom scores (r=0.52 and r=0.48, respectively) and negatively with the mean percent PEFR on waking (r=-0.50 and r=-0.65, respectively) recorded for 2 weeks prior to the sputum collection. These findings suggest that the eosinophil activation in the airway is closely linked to the symptoms and airflow obstruction of asthma, and that the ECP concentration in induced sputum could serve as useful marker for evaluating the severity of asthma and monitoring airway inflammation to achieve the optimal control of asthma.

Adolescent↗
PubMed1997

Subcutaneous tissue distribution of vancomycin from a fibrin glue/Dacron graft carrier.

We investigated the tissue distribution of vancomycin (VCM) incorporated in fibrin glue (FG) in a rat model. One VCM-loaded FG Dacron graft (VCM-FG, VCM 0.6 mg/ graft) was implanted in the subcutaneous tissue of the anterior abdominal wall of each rat. VCM was injected intravenously at an equal dose (0.6 mg/rat) after implantation of one control graft (without VCM-FG). After the implantation and the iv injection of an equal dose of VCM (0.6 mg/rat), the tissue distribution of VCM for up to 24 h was determined through analysis of the implanted VCM-FG grafts, which released VCM over a 24 h period. The area under the VCM concentration-time curve (AUC) of the tissue was 89.58 micrograms.h/g after the implantation of the VCM-FG graft, and 7.40 micrograms.h/g after the iv injection of VCM, respectively. The targeting index of the tissue, defined as the ratio of AUC after the implantation of the VCM-FG graft to that after VCM iv injection, was 12.11. None of the six VCM-FG Dacron grafts after implantation became infected following inoculation with S. aureus ATCC 25923 (0.1 mL 10(8) CFU/mL). These results suggest that this VCM-FG Dacron graft delivery may be useful in preventing local infection by enhancing the delivery of VCM to the local areas of the implanted site in rats.

Animals↗
PubMed1997

Hyperventilation as a specific test for diagnosis of coronary artery spasm.

The hyperventilation test has been used as a clinical tool to induce coronary spasm. However, its diagnostic and prognostic values have not been fully elucidated. This study was designed to establish the sensitivity and specificity of the hyperventilation test and to clarify the characteristics of hyperventilation test-positive patients. We examined 206 patients in whom coronary spasm was documented by angiography (spasm group), and 183 patients without angina at rest in whom acetylcholine failed to induce spasm (nonspasm group). All patients performed vigorous hyperventilation for 6 minutes in the early morning. Of the spasm group patients, 127 showed positive responses to the test, including ST elevation (n = 111), ST depression (n = 15) and negative U wave (n = 1). None in the nonspasm group showed any ischemic electrocardiographic change. Thus, the sensitivity and specificity of this test for diagnosis of coronary spasm were 62% and 100%, respectively. In the spasm group, there were no significant differences between hyperventilation test-positive and test-negative patients in age, sex, the prevalence of hypertension, diabetes mellitus, obesity, smoking, and the number of diseased vessels. When clinical characteristics were compared, the proportions of the patients with high disease activity (> or =5 attacks a week), with severe arrhythmias (second- or third-degree atrioventricular block and/or ventricular tachycardia) during attacks, and with multivessel spasm were significantly higher in the hyperventilation test-positive patients than in the negative patients (69% vs 20%, p <0.0001; 31% vs 11%, p <0.005; and 58% vs 34%, p <0.01, respectively). These findings imply that hyperventilation is a highly specific test for the diagnosis of coronary artery spasm, and that hyperventilation test-positive patients are likely to have life-threatening arrhythmias during attacks and multivessel spasm.

Adult↗
PubMed1997

Characterization of proviral DNA from an individual with long-term, nonprogressive infection with HIV-1 and nonrecoverable virus.

A small proportion of individuals infected with HIV-1 known as long-term nonprogressors (LTNPs) remain healthy and immunologically normal, with stable numbers of CD4+ lymphocytes, for prolonged periods without the administration of antiretroviral agents. The long terminal repeat (LTR) of HTV-1 proviral DNA of an LTNP from whom virus was consistently not recoverable has now been isolated by a nested polymerase chain reaction (PCR) method and shown to contain a total of 38 point mutations, only four of which affect promoter and enhancer elements, compared with the IIIB strain of HIV-1. Almost the entire HIV-1 proviral DNA was then isolated from the proband by a long PCR approach. Restriction enzyme digestion of the proviral DNA revealed no large deletions in the gag, pol, or env genes, although the loss of an Nco I site was apparent. Amplification of the env gene by long PCR also yielded a product apparently identical in size to that obtained with HIV-1 strain IIIB. Analysis by long PCR of HIV-1 proviral DNA from LTNPs with nonrecoverable virus may clarify the mechanism of long-term nonprogression and contribute to the development of HIV-1 vaccines.

Acquired Immunodeficiency Syndrome↗
PubMed1997

The extent of lymph node dissection for colon carcinoma: the potential impact on laparoscopic surgery.

BACKGROUND: The surgeon is no longer able to palpate the mesocolon for lymph node metastases during laparoscopic colectomy. The extent of lymph node dissection should be determined beforehand for cancer control. METHODS: The distribution of lymph node metastases was obtained by the clearing method on colon carcinomas for 164 patients. RESULTS: For pericolic spread: for pT1 tumors, the distance from the primary tumor to a metastatic lymph node was 2.5 cm; for pT2, the distance was within 5 cm; for 97.0 % of pT3 tumors with lymph node metastases, the distance was within 7 cm; for 93.3 % of pT4 tumors with lymph node metastases, the distance was within 7 cm. For central spread: for pT1 tumors, the rate of metastasis to central lymph nodes was 0 %; for pT2, the rate of metastasis was 20.0 % to intermediate lymph nodes; for pT3, the rate of metastasis was 30.6 % to intermediate lymph nodes and 15.3 % to main lymph nodes; for pT4, the rate of metastasis was 44.4 % to intermediate lymph nodes and 22.2 % to main lymph nodes. CONCLUSIONS: Central lymph node dissection is not required for patients with T1 carcinomas, but proximal and distal 3-cm margins of resection are required. For T2, central lymph node dissection that includes the intermediate lymph node should be performed, as well as 5-cm proximal and distal margins of resection. For T3 and T4, central lymph node dissection including the main lymph node should be performed, as well as 7-cm proximal and distal margins of resection. [See editorial on pages 177-8, this issue.]

Colectomy↗
PubMed1997

Demonstration of the facilitatory role of 8-OH-DPAT on cholinergic transmission in the rat hippocampus using in vivo microdialysis.

The role of the serotonin (5-HT)1A receptor in the regulation of acetylcholine (ACh) release in the hippocampus was investigated using an in vivo microdialysis technique and a sensitive radioimmunoassay specific for ACh. The mean (+/- S.E.M.) basal ACh contents in the hippocampal perfusate of conscious, freely moving rats was 60 +/- 4 (n = 29) and 3691 +/- 265 fmol/30 min (n = 31), respectively, in the absence and presence of physostigmine (Phy) in the perfusion fluid. Systemic administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.5 mg/kg, s.c.), a 5-HT1A agonist, significantly enhanced ACh release both in the presence and absence of Phy. Local application of 8-OH-DPAT (3-30 microM) into the hippocampus through the microdialysis probe significantly potentiated ACh release only in the presence of Phy, whereas no significant effect was observed in its absence. Pretreatment with NAN-190 (3 mg/kg, i.p.), a 5-HT1A antagonist, eliminated the increasing effect of systemically applied 8-OH-DPAT on ACh release, while NAN-190 alone had no effect on basal ACh release either in the absence or presence of Phy. Consistent with the time course of ACh release, systemic administration of 8-OH-DPAT evoked hyperlocomotion, which was reversed by NAN-190. However, local hippocampal application of 8-OH-DPAT did not affect the locomotor activity of the rats. These findings suggest that at least two different sites are involved in the 8-OH-DPAT-induced increase in the release of ACh in the rat hippocampus in vivo.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism.

Nitric oxide (NO) is an endothelium-derived relaxing factor and its main mechanism of action is activation of soluble guanylyl cyclase. NO and NO-related compounds have been reported to affect several neuronal functions in the central nervous system. In this study, we investigated the effects of NO donors (sodium nitroprusside (SNP) and (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409)) on acetylcholine (ACh) release from rat hippocampal slices. SNP (10(-5) M) and FK409 (10(-4) M) increased electrical stimulation-evoked ACh release without affecting basal release. As dibutyryl cyclic GMP inhibited stimulation-evoked ACh release, the effects of these NO donors were not due to soluble guanylyl cyclase activation. Atropine increased stimulation-evoked ACh release by blocking presynaptic muscarinic autoreceptors, and SNP increased stimulation-evoked ACh release in the presence of atropine, suggesting that SNP and atropine increase stimulation-evoked ACh release by different mechanisms. The present results indicate that NO enhances some part of the excitation-secretion coupling pathway without inducing ACh release directly and these effects are mediated by cyclic GMP-independent mechanism.

Acetylcholine↗
PubMed1997

Melanoma antigen-encoding gene-1 expression in invasive gastric carcinoma: correlation with stage of disease.

BACKGROUND: A human melanoma antigen-encoding gene-1, MAGE-1 gene, may be linked to the neoplastic transformation. In the present study, we extended this association with human gastric carcinomas. Specifically, we focused on the relationship between MAGE-1 gene expression and the histologic stage of gastric carcinoma. METHODS: We used a reverse transcription-polymerase chain reaction assay (RT-PCR) to analyze the expression of the MAGE-1 gene in 38 endoscopic biopsy specimens from gastric carcinomas. We also studied the relationship between the expression of MAGE-1 gene and the genetic expression of several tumor invasion-related factors, including 72 kD type IV collagenase (MMP2), urokinase-type plasminogen activator (uPA), platelet-derived growth factor A (PDGF-A), and vascular endothelial growth factor (VEGF). RESULTS: Eleven of the 38 tumor samples (28.9%) expressed the MAGE-1 gene. MAGE-1 gene expression was present only in two of the 38 adjacent nontumor samples (5.3%). MAGE-1 gene expression in the 38 tumor samples was significantly correlated with the histological stage of disease (P = 0.0008), especially with the depth of histologically confirmed tumor invasion (t1 vs. t2 or greater, P = 0.00048). The expression of MAGE-1 gene correlated with the expression of MMP2 (P = 0.0064), uPA (P = 0.0390), and PDGF-A (P = 0.00018). CONCLUSIONS: These data suggest that the MAGE-1 gene may be activated in gastric carcinomas during periods of their development or invasion. In addition, a relationship between MAGE-1 gene expression and expression of invasion-related factors has been demonstrated.

Antigens, Neoplasm↗
PubMed1997

Ex vivo and in vivo evaluation of the blood compatibility of surface-modified polyurethane catheters.

Catheter model tubes were prepared from a medical-grade polyetherurethane and their outer surfaces modified by surface-graft polymerization of acrylamide and dimethyl acrylamide (DMAA). The surface-graft layer was characterized by means of dry staining, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, and protein adsorption. Ex vivo evaluation for the blood compatibility of the surface-modified polyurethane was carried out using the polyurethane tube as an arterio-venous shunt between the carotid artery and the jugular vein of rabbits. When the surface density of grafted polymer was in the range of 10-30 microg/cm2, the in vitro adsorption of IgG exhibited a minimum value and platelet adhesion to the grafted polyurethane surface was insignificant, in marked contrast with that to the virgin (nonmodified) surface. The in vivo blood compatibility of polyurethane was evaluated by implanting the catheter tube in the inferior vena cava of rabbits from the femoral vein after ligation of a distal site of the exposed femoral vein. After remaining there for predetermined periods of time, the implanted catheters were taken out together with the veins of the rabbits that had been heparinized and sacrificed just prior to excision of the veins. After exchange of the blood in the veins for saline, the excised veins were opened by cutting longitudinally to inspect for clot formations on the surfaces of the implanted catheters. Occlusion of the inferior vena cava was not observed for any of the catheters, nor was there any apparent damage or microembolizations in the lungs and kidneys. Many small-sized clots were observed on the surfaces of the nonmodified polyurethane tubes after a 2-week implantation whereas the catheter surfaces grafted with DMAA polymer chains had a much smaller number of clots. When the blood compatibility of polyurethane surfaces was graded for relative evaluation from one (marked clotting) to five (no clotting) based on the size and number of the clots, the evaluation results were as follows: 3.1 (virgin, 2 weeks), 4.0 (grafted, 1 week), 4.1 (grafted, 2 weeks), and 3.5 (grafted, 1 month).

Acrylamides↗
PubMed1997

Ischemia-reperfusion injury on the pancreas in rats: identification of acinar cell apoptosis.

An ischemia-reperfusion injury on the pancreas is involved in the pathophysiology of acute pancreatitis or tissue injuries after pancreas transplantation. On the other hand, recent studies have demonstrated that ischemia-reperfusion induces apoptosis in several organs such as kidney, heart, and brain. In the present study, we sought to characterize a pattern of injury during ischemia-reperfusion on the pancreas and determined whether ischemia-reperfusion on the pancreas causes the apoptotic process. Ischemia-reperfusion was induced by blocking the inferior splenic artery and removing the clamp in pentobarbital-anesthetized rats. Rats were sacrificed at 0-72 hr following a 60-min ischemia. Evans blue extravasation showed 3.5-fold increase at 2 hr after reperfusion, indicating a rapid increase of vascular permeability. Tissue myeloperoxidase activity, an index of neutrophil accumulation, significantly increased in a time-dependent manner until 48 hr after reperfusion. Histological analysis revealed the existences of interstitial cell infiltration and edema. DNA breaks of acinar cells were detected by gel electrophoresis and in situ nick end-labeling, and the numbers strikingly increased at 48 hr after reperfusion. Furthermore, Bax protein, an effector of apoptotic cell death, was expressed in acinar cells. The results indicate that an ischemia-reperfusion injury on the pancreas in rats resembles many features of acute pancreatitis. Apoptosis in acinar cells may be one of the specific features of the ischemia-reperfusion injury on the pancreas.

Amylases↗
PubMed1997

Detection of matrix metalloproteinase activity in human pancreatic cancer.

Destruction of the basement membrane (BM) is mandatory for tumor spread, and matrix metalloproteinases (MMPs) are known to be implicated in colon cancer invasion and metastasis by digesting type IV collagen, a main component of the BM. The current study analyzed the expression of MMP-2 and MMP-9 in pancreatic cancer tissues. Frozen specimens of pancreatic cancer (n = 10), a liver metastatic nodule from pancreatic cancer (n = 1), and normal pancreas (n = 3) were homogenized and analyzed by zymography. The activated form of MMP-9 (82 kDa) was detected in all of the normal and malignant tissues, while the activated form of MMP-2 (62 kDa) was detected in all of the pancreatic cancers and its metastatic tissue, but not in the normal pancreatic tissues. These results indicate that expression of the activated form of MMP-2 may be specific to pancreatic cancer, while that of MMP-9 may be unrelated to it.

Biomarkers, Tumor↗
PubMed1997

Endoscopic ultrasonography for demonstrating loss of multiple-layer pattern of the thickened gallbladder wall in the preoperative diagnosis of gallbladder cancer.

The purpose of this study was to elucidate the roles of endoscopic ultrasonography (EUS), conventional US, CT, and MRI in differential diagnosis of gallbladder wall thickening. We scrutinized images for the presence of the multiple-layer patterns of the thickened gallbladder walls during preoperative images (EUS, n = 22; US, n = 23; CT, n = 20; MRI, n = 15) and retrospectively correlated them with surgical results in 25 patients. The pathological diagnoses included 7 gallbladder cancers, 9 cases of chronic cholecystitis, 5 cases of xanthogranulomatous cholecystitis, and 4 cases of adenomyomatosis. Multiple-layer patterns of gallbladder wall were observed in patients with inflammatory and benign diseases by US, EUS, CT, and MRI. This pattern was demonstrated by EUS more efficiently compared with other means of imaging. All subjects with loss of multiple layers were finally diagnosed by use of EUS as having gallbladder cancer at surgery. Loss of multiple-layer patterns of the gallbladder wall demonstrated by EUS was the most specific finding in diagnosing gallbladder cancer.

Adenomyoma↗
PubMed1997

A simple and reliable quick-freezing/freeze-fracturing procedure.

We describe a simple method for the quick-freezing/freeze-fracturing of cells in tissues or culture monolayers. Tissue slices or cultured cells were covered with thin copper foil (10-micron-thick), and frozen by smashing them against a liquid helium-cooled copper block. Freeze-fracturing was accomplished by mechanically separating the copper foil from the frozen specimen. The fracture faces were replicated by platinum and carbon. Replicas were processed for conventional electron microscopic observation or cytochemical labeling. This method allows the ultrastructural and cytochemical examination of large areas of fractured membrane without chemical fixation.

Animals↗
PubMed1997

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application.

Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy.

Animals↗
PubMed1997

Increased hepatic beta-oxidation of docosahexaenoic acid, elongation of eicosapentaenoic acid, and acylation of lysophosphatidate in rats fed a docosahexaenoic acid-enriched diet.

Rats were fed a diet supplemented with corn oil (n-3 deficient), soy oil, or a mixture containing 8% 22:6n-3 ethyl ester for 6 wk. The hepatic capacities for the beta-oxidation and synthesis of 22:6n-3, in addition to the acylation of lysophosphatidate, were tested in vitro. In rats that were fed a 22:6n-3-enriched diet, both the beta-oxidation of 22:6n-3 and elongation of 20:5n-3 were enhanced compared to those in rats fed the other diets. Acylation of lysophosphatidate was also enhanced in rats fed a 22:6n-3-enriched diet, while the rate of dephosphorylation of phosphatidate was not changed. The amount of 22:6n-3 in the liver was much less than that consumed in a docosahexaenoic acid-enriched diet. These results suggest that a significant amount of dietary 22:6n-3 was degraded via beta-oxidation, and that a portion of the retroconverted 20:5n-3 was recycled for the synthesis of 22:6n-3. The recycling of 20:5n-3 might contribute to the low level of 22:6n-3 in rats fed an n-3-deficient diet.

Acylation↗