Genotoxicity tests. Considerations regarding the selection of a standard battery test.
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Animals↗
Biomedical subjects
H Shimada
Publications and source records attributed to H Shimada.
Clinical application of a novel DNA fluorescence intercalator, pyrylium dye, for the quantification of PCR products.
Polymerase chain reaction (PCR) is a powerful tool for clinical diagnosis in the field of oncology, infection, and allergy. However, a simple and sensitive method for quantifying PCR products has not been established. We therefore used a novel fluorescence DNA intercalator, pyrylium iodide (P2) to quantify PCR products. A 838-bp fragment of the human beta-actin gene and a 374-bp fragment of the gene for ornithine decarboxylase (ODC) and ODC primer dimers were generated by PCR and quantified using either P2 or another fluorescence DNA intercalator, YOYO-1. In addition, utilizing RT-PCR and the P2 method, the ODC mRNA expression from 10 colonic cancers was quantified. Serially diluted beta-actin and ODC PCR products could be quantified using P2 without having to separate them from primer dimers and other components of the reaction mixture. However, YOYO-1 could not be used to quantify the PCR products because of high background fluorescence from the primer dimers. In the clinical study, ODC mRNA expression as quantified by P2 was significantly higher in cancerous tissue (113.8 +/- 4.1; mean +/- SEM; plate reader units) than in mucosa of normal appearance (68.4 +/- 4.8). P2 is a promising tool for quantifying PCR products.
Actins↗
Amyloid beta protein deposition in normal aging has the same characteristics as that in Alzheimer's disease. Predominance of A beta 42(43) and association of A beta 40 with cored plaques.
Two distinct species of amyloid beta protein (A beta) with different C-termini, A beta 42(43) and A beta 40, are deposited in senile plaques (SP) of Alzheimer's disease (AD), with the former being far predominant. To investigate whether A beta 42(43) also predominates over A beta 40 in normal aging, we examined by immunocytochemistry the C-termini of A beta in SP in the brains of non-demented aged individuals and compared the results with those in AD. Virtually all SP were A beta 42(43)-positive; of these 12% in non-demented aged individuals and 25% in AD patients (mean of three areas examined) were also A beta 40-positive. In both the AD and non-demented groups, 2/3 of the A beta 40-positive SP were typical cored SP. These results indicate that A beta 42(43) is the predominant species deposited in SP in normal aging, and there is no qualitative difference in terms of the C-terminus of A beta in the parenchymal amyloid deposition between normal aging and AD.
Aged↗
[Prospective randomized trial comparing 1/2 FAM (5-fluorouracil (5-FU) + adriamycin + mitomycin C) versus palliative therapy for the treatment of unresectable pancreatic and biliary tract carcinomas (the 2nd trial in non-resectable patients). Japanese Study Group of Surgical Adjuvant Therapy for Carcinomas of the Pancreas and Biliary Tract].
The efficacy of 1/2 FAM, which consists of 5-fluorouracil (5-FU), adriamycin (ADM) and mitomycin C (MMC), was compared with that of palliative treatment in patients with unresectable pancreatic and biliary tract carcinomas in a multicenter randomized trial. The patients assigned to 1/2 FAM group were treated with 5-FU 200 mg/m2/day IV, ADM 15 mg/m2/day IV and MMC 5 mg/m2/day IV. These 3 drugs were given concurrently as the initial dose within a week after palliative operation, and this regimen was repeated for at least 2 whole courses, at 4-week intervals before the next course of therapy. Those randomized to the control group were subjected to palliative treatment alone. Completely eligible for analysis were 42 cases of the 1/2 FAM group and 41 of the control group. There was no significant difference between the groups with respect to the overall and differentiated survival times according to the tumor sites and the clinical efficacy. As for the duration of 50% inhibition of tumor progression, a significantly better outcome was obtained in 1/2 FAM group. Tumor progression was most significantly inhibited in patients with gallbladder carcinoma. In 1/2 FAM group, tumor reduction was achieved in 1 CR and 2 PR patients. The most frequent adverse reaction was gastrointestinal manifestations, along with diarrhea and alopecia. 1/2 FAM did not contribute to the life prolongation, but inhibited the tumor progression for a significantly longer duration and, to a lesser extent, reduced the tumor size in unresectable pancreatic and biliary tract carcinomas. This regimen is suggested to be useful particularly in the treatment of the latter carcinoma.
Adult↗
Miniature swine MHC class II heterodimers expressed through double-copy retroviral vectors.
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Animals↗
[Prognostic factors of colorectal cancer concerning metastases].
Four prognostics factors were investigated for colorectal cancer metastases. 1) There were statistically more venous invasions using Victoria blue elastic staining in patients with liver or lymph node metastasis than in those without metastasis. 2) The immunohistochemical expression rate of c-erbB-2 in liver metastasis cases was 27%, which was significantly higher than 3% in no metastasis cases. 3) Sialyl Lewis x (SLex) is related with cell adhesion. SLex positive rates in vessel invasion cancer cells were 71.4% with metastasis and 31.0% without metastasis. 4) Matrilysin is one of MMPs and it was significantly increased with Dukes stage by fluorescence intensity.
Colorectal Neoplasms↗
[An unusual autopsy case of corticobasal degeneration--with special reference to clinicopathological differentiation from progressive supranuclear palsy and slowly progressive aphasia].
We report an unusual autopsy case of corticobasal degeneration. The patient was male who was 67 years old at the time of his death. He developed clumsiness of his right hand at age 65. Neurological examination at age 66 revealed supranuclear ophthalmoplegia of upward gaze and parkinsonism. Progressive supranuclear palsy was suspected. The patient's clinical course was rapidly progressive. Dementia developed nine months after the onset of the disease, and the patient manifested apallic syndrome at 21 months and died of pneumonia at about 25 months. At autopsy the brain weighed 1370g. Macroscopic examination revealed prominent atrophy of the anterior and parietal lobes, particularly of the pars opercularis in the left inferior frontal gyrus, and there was marked depigmentation of the substantia nigra. Histological examination showed neuronal loss, glial proliferation, spongy state, and ballooned neurons in the frontal and parietal lobes. Neuronal loss with glial proliferation was conspicuous in the striatum, pallidum, thalamus, and substantia nigra. There was slight neuronal loss in the dentate nucleus and locus ceruleus. Staining by Gallyas-Braak method revealed massive appearance of argyrophilic threads. This case is important in terms of the clinical differentiation from progressive supranuclear palsy, and is also interesting in regard to the pathological correlation with slowly progressive aphasia.
Aged↗
[A study of the significance of serum beta 2-microglobulin levels in patients with multiple myeloma--analyzes as a marker of renal dysfunction and as a marker of tumor cell mass].
Renal involvement known as a myeloma kidney is often observed in patients with multiple myeloma (MM). This complication has been recognized as one of the most important prognostic factors in this disease. Serum beta 2-microglobulin (S beta 2-m) has been also recognized one of the prognostic factor that reflects both a glomerular infiltration rate and a volume of neoplastic cells, because beta 2-m usually can be produced by the neoplastic lymphoid cells. To clarify the significance of the S beta 2-m in MM, we compared S beta 2-m levels and the clinical stage, with another clinical parameters for renal function such as 24 hr creatinine clearance (24 hr Ccr), N-acetyl-beta-glucosaminidase (NAG) levels in urine and serum alpha 1-microglobulin (S alpha 1-m) levels. The elevated S beta 2-m levels were commonly observed not only in patients with stage IIIB, but also in stage IA, IIA and IIIA (76%) who have normal renal function judged by the serum nitrogen or creatine levels. S beta 2-m levels correlated with 24 hr Ccr the most, then correlated with S alpha 1-m levels and less correlated with NAG levels in urine. Although a single elevation of S beta 2-m levels with other normal findings of renal parameters was found only in three out of 30 MM patients, the S beta 2-m levels in these patients did not change after the chemotherapy which had led to the diminution of serum M-protein. Together with these results, it was suggested that the S beta 2-m levels mainly reflect the renal dysfunction even if it stays in the subclinical stage, and reflect less the number of neoplastic cells in MM patients.
Biomarkers↗
Mechanism of stereoselective serum binding of ketoprofen after hemodialysis.
In vitro studies of serum protein binding were conducted to examine the influence of hemodialysis therapy on the stereoselectivity for ketoprofen (KP) in patients with renal dysfunction. In normal volunteers, only small differences in binding to undiluted serum were observed between the enantiomers of racemic KP (10 micrograms/mL). As compared with healthy volunteers, significantly impaired binding to undiluted serum was observed in uremic patients before they received dialysis. This reduced binding was not accompanied by alterations in stereoselectivity. At the end of dialysis, a similar extent of defective binding to undiluted serum was still observed, whereas interestingly, its stereoselectivity was significantly enhanced. These findings indicated that preferential inhibition for S-enantiomer serum binding was caused by changes that occurred during hemodialysis. Among various physiological parameters, only enantioselective inhibition of KP serum binding was correlated with heparin-induced increases in free fatty acids (FFA) levels and with unbound levels of uremic toxins with indole ring, indoxyl sulfate and indole acetate. To elucidate the effects of these accumulated endogenous substances in serum of patients, ultrafiltration was also used to study the binding of KP enantiomers to isolated human serum albumin under various conditions. After dialysis, mean albumin level and pH value in patients were slightly but significantly altered, but in vitro binding data indicated that these factors could clearly be ruled out as an explanation for the origin of the induced stereoselectivity. In contrast to palmitate, oleate and laurate markedly induced the stereoselective inhibition, which indicated that these fatty acids preferentially inhibited the binding of S-KP via a competitive and allosteric mechanism. Among the four uremic toxins, only indoxyl sulfate and indole acetate predominantly displaced the S-enantiomer rather than its antipode through competitive interaction. In addition, serum unbound levels of indole uremic toxins were correlated with elevated FFA levels. FFA displaced those indole uremic toxins in a manner similar to KP, which suggests that FFA also indirectly inhibits the S-enantiomer binding by increasing the unbound levels of indole uremic toxins. Thus it is likely that combined direct and cascade effects of transiently increased FFA and unbound indole uremic toxins contributed to the preferential elevation of the pharmacologically active S-KP fraction in uremic patients at the end of hemodialysis.
Adult↗
Laboratory diagnosis of Helicobacter pylori infection by polymerase chain reaction.
Although the detection of Helicobacter pylori (Hp) infection is important, it is a rather difficult task using only conventional diagnostic techniques. In this study, we have applied a polymerase chain reaction (PCR)-based assay for the diagnosis of Hp infection. RT-PCR for Hp ribosomal RNA was utilized, using the gastric biopsy specimens obtained endoscopically from 10 patients. PCR primers have been designed from the highly consensus region of the 7 different Hp rRNA sequences in GenBank. In the detection of Hp, PCR was more sensitive than culturing and showed 100% of the sensitivity and specificity compared with histology. Our primer pair could detect as few as 50 organisms per reaction. RT-PCR for Hp rRNA is a sensitive and specific method to detect Hp infection in the gastric mucosa.
Base Sequence↗
Suppression of c-jun by antisense oligonucleotides inhibits cell adhesion but not respiratory burst during phorbol ester-induced differentiation of U937 human monoblastic cells.
We studied the role of the immediate early gene c-jun in cell proliferation and phorbol 12-myristate 13-acetate (PMA)-induced differentiation in U937 human monoblastic cells, using c-jun-specific antisense (AS) phosphorothioate oligonucleotides. In selecting the most specific and potent oligonucleotide sequence, we performed extensive analyses for the binding specificity between all candidates of c-jun AS oligonucleotides and the whole sequences in GenBank database, using a computer program. Among the 20 selected oligonucleotides, two potent 15-mer AS oligonucleotides (C-JUN AS oligonucleotides) exhibited significant inhibition of cell growth in a dose-dependent manner between 2 and 10 microM. Reverse transcription-PCR and Western blot analysis demonstrated that 10 microM of C-JUN AS oligonucleotides reduced c-jun expression at both the mRNA and protein levels. More importantly, C-JUN AS oligonucleotides showed distinct effects on two markers of PMA-induced differentiation; the C-JUN AS oligonucleotides inhibited cell adhesion, whereas they did not affect another marker of differentiation, respiratory burst (measured by nitro blue tetrazolium reduction assay). These results suggest a critical role of c-jun in both cell proliferation and PMA-induced cell adhesion but not in PMA-induced respiratory burst in U937 cells.
Base Sequence↗
[Probability of vaccine therapy for pancreas cancer].
To explore the possibility of vaccine therapy for pancreatic cancer using ras oncogenic protein related antigen we studied whether cytotoxic T lymphocyte for tumor rejection can be induced by vaccination using the peptide relating mutated ras protein. Splenocytes from mice immunized by peptide (9-15 mer) relating mutated ras protein with normal splenocytes specifically increased in the culture with the peptide. The harvested splenic lymphocytes from the mice had peptide specific lysis against B 6 fibroblast incubated with the peptide. These results suggested that immunization using the peptide relating mutated ras peptide with autologous antigen presenting cell can be a vaccine therapy for pancreatic cancer.
Animals↗
Fluorometric determination of total mRNA with oligo(dT) immobilized on microtiter plates.
We have developed a rapid and nonradioactive method of quantifying cytosolic mRNA from crude cell lysates by using plastic plates to which oligonucleotides containing poly(dT) sequences were previously immobilized. Captured mRNA on the plate was mixed with Yoyo-1 fluorescent indicator dye, and the resulting Yoyo-1 fluorescence of the mRNA-Yoyo-1 complex was measured in a fluorometer. Because Yoyo-1 signals were linearly increased in proportion to the amount of applied mRNA in the range 10-250 ng, the amount of mRNA in test samples can be determined by comparing their Yoyo-1 fluorescence with that of known concentrations of calibrator mRNA. Using this system, we found that the amount of cytosolic mRNA in undifferentiated U937 and HL-60 cells was 268.6 +/- 13.1 and 282.0 +/- 7.8 ng/10(6) cells, respectively, significantly (P < 0.01) more than that of phorbol ester-induced differentiated U937 and HL-60 cells (145.3 +/- 13.9 and 164.7 +/- 11.6), respectively. Therefore, the present system may be applicable to both medical molecular biology research and diagnostics.
Animals↗
Thoracoscopic lung resection for lung metastasis of colorectal cancer.
We performed thoracoscopic lung resection seven times in four patients with lung metastases from colorectal cancer. This procedure allowed a short operation time, minimal blood loss, and a short hospital stay. Thoracoscopic lung resection was chosen for resection of lung metastases from colorectal cancer because it allowed simultaneous bilateral lung resection and early postoperative adjuvant chemotherapy.
Adult↗
[A study about the mechanism of hepatocyte injury induced by endotoxin--interaction of Kupffer cells and polymorphonuclear neutrophils (PMNs)].
Focusing our attention on the TNF (tumor necrosis factor) produced by Kupffer cells (KCs). The role of KCs and polymorphonuclear neutrophils (PMNs) in endotoxin (LPS)-induced hepatocellular injury was investigated. This study used the culture fluid supernatant of KCs which had been stimulated with LPS as the "LPS stimulation supernatant", and evaluated hepatocellular injury as ornithine carbamyl transferase percent leakage. There was no difference between groups I (HCs: hepatocytes) and II (HCs + PMNs), but there were differences between groups I and III (HCs + LPS stimulation supernatant, groups II and IV (HCs + LPS stimulation supernatant + PMNs), and III and IV. There was no significant difference between III and V (III + anti-TNF antibody), but a difference was found between IV and VI (IV + anti-TNF antibody). These findings suggest that PMNs activated by KCs-generated TNF as well as KCs-derived humoral factors other than TNF play a role in the development of LPS-induced hepatocellular injury.
Animals↗
Endoscopic screening for esophageal cancer in 788 patients with head and neck cancers.
Esophageal cancer has a poor prognosis because it is difficult to detect in its early stages and, even if an operation is possible, the postoperative quality of life is much impaired. An early diagnosis can lead to a good prognosis and enables treatment by endoscopic mucosal resection (EEMR), contributing to a postoperative good quality of life. As head and neck cancers are known to have a high risk of concomitant esophageal cancer, endoscopic screening with iodine staining was performed on 788 patients with head and neck cancers. Among them, 93 cases of esophageal cancers (11.8%) and 23 cases of gastric cancers (2.9%) were detected. Seventy-two cases (77.4%) of the 93 esophageal cancers were superficial cancers limited to the submucosal layer. Twenty cases, treated by EEMR, had a good postoperative course without local recurrence. We suggest that endoscopic screening for esophageal cancer should be performed on all patients with head and neck cancers, because it allows early detection and a good prognosis, and the treatment can be completed by endoscopic maneuver.
Aged↗
Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence.
p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.
Amino Acid Sequence↗
Effects of chelating agents on testicular toxicity in mice caused by acute exposure to nickel.
N-Benzyl-D-glucaminedithiocarbamate (BGD), diethyldithiocarbamate (DDTC), dihydroxyethyldithiocarbamate (DHED), trans-1,2-cyclohexanediamine N,N,N',N'-tetraacetic acid (CDTA), and meso-2,3-dimercaptosuccinic acid (DMSA) were studied for their protective effects against the testicular toxicity in mice induced by acute exposure to nickel (Ni). Mice were injected intraperitoneally with NiCl2 (5 mgNi/kg) and 30 min or 24 h later, they were injected intraperitoneally with chelating agents (400 mumol/kg). Ni injection increased lipid peroxidation and concentrations of Ca and Fe in the testes, liver, and kidney, and decreased the testicular weight and the fertility rate. At 30 min after Ni treatment, the chelating agents other than CDTA effectively depressed Ni concentration in the testes. At 24 h after Ni treatment, DMSA, BGD, and DDTC were effective in mobilizing Ni from the testes. DMSA, BGD, and CDTA significantly prevented the increase in the lipid peroxidation, the increase in the concentrations of Ca and Fe in the testes, liver, and kidney, and the decrease in the fertility rate caused by Ni injection. Treatment with DMSA or BGD was more effective than that with the others in decreasing the testicular Ni concentration, resulting in effective protection against Ni-induced testicular damage.
Animals↗