Immunocytochemical detection of active DNA synthesis by in vivo labelling with anti-bromodeoxyuridine antibodies in glomeruli of rats with nephrotoxic serum nephritis

We studied DNA synthesis in rats with nephrotoxic serum nephritis (NTSN), a model of a glomerular disease, using in vivo labelling with 5-bromo-2'-deoxyuridine (BrdUrd). NTSN was induced by intravenous injection of subnephritogenic doses of rabbit anti-rat GBM antiserum into male Sprague-Dawley rats. Each rat received a single injection of the DNA precursor, 3H-thymidine analog (BrdUrd), ten min before the tissues were removed. For immunocytochemical detection of DNA synthesis, semithin sections were prepared at various intervals (4 h up to 84 days) after pulse labelling. Using a monoclonal anti-BrdUrd antibody, BrdUrd-incorporated DNA-synthesizing cells were noted in the proliferative zone of the gastric mucosa at all times. In NTSN, BrdUd incorporated DNA-synthesizing cells were detected in the glomeruli from 4h through 28 days after inoculation, with the peak occurring at days 2 to 4. On those days, up to half of the glomeruli showed BrdUrd-incorporated cells, with 8 cells per glomerulus as a maximum. From days 7 to 28, few glomerular cells incorporated BrdUrd, and none did so after day 28. The majority of the BrdUrd-incorporated cells were endothelial. These results suggest that active DNA synthesis by glomerular endothelial cells occurs during a short period of the heterologous phase in this model, and that the lack of mesangial cell proliferation might explain the self-limiting nature of this model. By using in vivo labelling with BrdUrd, we were also able to easily and accurately detect active DNA synthesis without consideration of the normal cell renewal.(ABSTRACT TRUNCATED AT 250 WORDS)     

A case of recurrent breast cancer responding to combination therapy with mitoxantrone (MIT), 5'-deoxy-5-fluorouridine (5'-DFUR) and medroxyprogesterone acetate (MPA)

The patient was a 50-year-old woman who had undergone left standard radical mastectomy who had undergone left standard radical mastectomy on June 1, 1986. She showed multiple liver metastases with elevation of CEA level in July, 1991, and 5'-DFUR plus MPA combination therapy was started. The daily dosages were: 800 mg/body and 1,200 mg/body, respectively. After intra-arterial infusion of pirarubicin and Lipiodol, bilateral oophorectomy was performed and an implantable reservoir for intra-arterial infusion chemotherapy was implanted via the proper hepatic artery. Then she was treated by arterial-infusion of mitoxantrone 10mg/body intermittently every two weeks. The metastatic foci responded to this therapy and her CEA level decreased.     

Thermal conductivity of five normal alkanes in the temperature range 283–373 k at pressures up to 250 MPa

Experimental data on the thermal conductivity of five liquid n-alkanes-hexane, heptane, octane, decane, and dodecane-are presented in the temperature range from 283 to 373 K at pressures up to 250 MPa or the freezing pressures. The measurements were performed on an absolute basis by an automated transient hot-wire apparatus. The uncertainty of the reported data is estimated to be within ±1%. The thermal conductivity of each alkane decreases almost linearly with rising temperature at a constant pressure and increases with increasing pressure at a constant temperature. Both the temperature coefficient of the thermal conductivity ¦(/T) _p¦ and the pressure coefficient (/P) _T decrease with increasing carbon number of alkanes. The experimental results were correlated with temperature and pressure by a similar expression to the Tait equation. It is also found that both the dense hard-sphere model presented by Menashe et al. and the modified significant structure theory proposed by Prabhuram and Saksena provide good representations of the present experimental results.     

Electropermeabilization in bladder cancer chemotherapy

PURPOSE: Electropermeabilization has been used for the introduction of genes into cells. Using this technique, we introduced the cytotoxic drug bleomycin (BLM) into cells and examined whether the technique might be useful for the treatment of bladder cancer. MATERIALS AND METHODS: For electropermeabilization in vitro, we used YTS-1 cells, a human transitional cell carcinoma line. Aliquots of cell suspension were mixed with a solution of BLM and immediately exposed to electric pulses. A high-power pulse generator was used to supply square-shaped pulses of 1250 V/cm (100 micros, eight pulses). After a 2-h post-shock incubation, cells were washed and incubated for one further hour. Then the concentration of BLM in the cells was measured using a bioassay. For electropermeabilization of tissue, we used normal male Wistar rats. The bladder was exposed and 10 mg/kg BLM was injected into the caudal vein. A series of eight pulses with a time constant of 100 micros at an electric field intensity of 1000 V/cm was applied. The bladder, liver and lungs were extracted 1 h later and prepared for quantification of the BLM concentration using the bioassay. RESULTS: Electrotreated cells contained significantly higher concentrations of BLM than nonelectrotreated cells. The concentration of BLM 1 h after electrotreatment in bladder tissue was 2.7 times higher than that in nonelectrotreated bladder tissue. CONCLUSION: The electropermeabilization technique has the potential to serve as a new and effective modality for the treatment of bladder cancer.     

Analysis of parent of origin specific DNA methylation at SNRPN and PW71 in tissues: implication for prenatal diagnosis

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental disorders caused by absence of paternal or maternal contributions of the chromosome region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting mutations. Molecular cytogenetic diagnosis is currently performed using a combination of fluorescence in situ hybridisation (FISH), DNA polymorphism analysis, and DNA methylation analysis. Only methylation analysis will detect all three categories of PWS abnormalities, but its reliability in tissues other than peripheral blood has not been examined extensively. Therefore, we examined the methylation status at the CpG island of the small nuclear ribonucleoprotein associated polypeptide N (SNRPN) gene and at the PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n = 48), amniotic fluid (AF) cell cultures (n = 25), cultured chorionic villus samples (CVS, n = 17), and fetal tissues (n = 18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, 20 LB cell lines, three AF cultures, one CVS, and 15 fetal tissues had been previously diagnosed as having deletions or UPD by other molecular methods. Methylation status at SNRPN showed consistent results when compared with FISH or DNA polymorphism analysis using all cell types tested. However, the methylation pattern for PW71 was inconsistent when compared with other tests and should therefore not be used on tissues other than peripheral blood. We conclude that SNRPN, but not PW71, methylation analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families known to carry imprinting centre defects.     

Pancreas and islet transplantations

Pancreas transplantation has been established as a treatment option for type I diabetes mellitus with one-year patients survival rate of 91% and one-year graft survival rate of 71%. Simultaneous pancreas and kidney transplantation with the bladder-drainage technique is most frequently performed. The bladder drainage technique makes amylase activity measurement in the urine as well as urine cytology possible, which facilitate a diagnosis of acute rejection. Combination treatment with cyclosporine, azatioprine, steroid and anti-lymphocyte globulin is usually employed for immunosuppression. In addition, FK506 in now available and expected to contribute to better graft survival. In contrast, islet transplantation has not yet achieved satisfactory results. Although a large number of islets can now be obtained from one pancreas, they are not sufficient for stabilizing a diabetic condition and multiple donors are still required. Xeno-transplantation may resolve the problem. Both pancreas and islet transplantation will achieve better results with further advance of transplant techniques including immunosuppressive treatment and diagnostic methods for acute rejection.     

Impaired hyperemic response of forearm vessels in patients with coronary artery disease. A non-invasive evaluation

The blood flow velocity of the right brachial artery was measured noninvasively by pulsed Doppler flowmetry in 50 patients with angina pectoris. Reactive hyperemia was induced by a 2-minute occlusion of the artery by a tourniquet. We assessed the peak velocity ratio (PVR) and 50% recovery time (RT) which were defined as the ratio of maximal to baseline systolic peak velocity and as the interval from the resumption of arterial flow to 50% decline of the increased systolic peak velocity, respectively. Multiple regression analysis for determinants of PVR and 50% RT was performed with 7 variables which were age, sex, hypertension, diabetes mellitus, smoking, total cholesterol level, and the number of diseased coronary arteries. Multiple R was 0.649 (p < .01) for PVR and 0.682 (p < .01) for 50% RT. There were significant inverse correlations between PVR and the number of diseased vessels (t-value; -3.34), hypertension (-2.43) and smoking (-2.38). The 50% RT was inversely correlated with the number of diseased vessels (t-value; -4.45), feminine gender (-2.75) or smoking (-2.12). Stepwise regression analysis revealed that the number of diseased vessels was the only significant variable for the determination of PVR or 50% RT. An impairment of reactive hyperemia at the forearm vessel correlated with the severity of coronary artery disease in patients with angina pectoris. This finding suggests the presence of some identical mechanisms which are detrimental to both vascular beds. Observation of the hyperemic response at the brachial artery will provide a clue for noninvasive estimation of the extent of coronary artery disease.     

Characteristics of acrylic acid and N-isopropylacrylamide binary monomers–grafted polyethylene film synthesized by photografting

Method for introducing grafted chains consisting of two types of monomer components, acrylic acid (AA) and N-isopropylacrylamide (NIPAAm), into low-density polyethylene (PE) film (thickness = 25 μm) was investigated by two photografting technique using xanthone photoinitiator at 60°C. In the first method (one-step method), AA and NIPAAm binary monomers were graftcopolymerized onto PE film. In the second method (two-step method), AA was first photografted onto PE film and then NIPAAm was further introduced into the AA-grafted PE film by a second-step photografting. Water absorbencies of the grafted films (one- and two-step samples) prepared by the one- and two-step methods, respectively, decreased in the order of AA-grafted film > one-step sample > two-step sample > NIPAAm-grafted film. The water absorbency steeply decreased at 20 to 40°C with increasing temperature when immersed in water at the temperatures (5–60°C) for 24 h. Thermosensitivity, which was defined as the ratio of water absorbencies of the grafted samples at 5 and 60°C, was higher for the one-step sample than the two-step one. The different extent of the water absorbency and the thermosensitivity between both samples is discussed in terms of location of grafted chains in the film substrate, which was determined by electron probe microanalysis and attenuated total reflection–infrared measurements, and monomer sequence distribution of the grafted chains. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67:2057–2064, 1998     

A few physical properties of grafted polyethylene and nylon films

Methyl methacrylate, vinyl acetate, or N-vinylpyrrolidone was graft-copolymerized onto low-density polyethylene or nylon 6 film by simultaneous irradiation method. Higher extent of grafting was attained by liquid-phase grafting than by vapor-phase grafting. The relative change in the degree of crystallinity of backbone polymer caused by grafting was measured with differential scanning calorimetry. In polyethylene-g-vinylpyrrolidone, the degree of crystallinity decreased markedly with the extent of grafting. However, in other grafted films, especially in grafted nylon, the crystallinity decrease was limited, or substantially no decrease was observed in a few systems. Water vapor permeability of the grafted films changed uniformly with increasing extent of grafting, and particularly polyethylene- and nylon-g-vinylpyrrolidone films showed excellent permeability, and some were able to be used as dialyzer. The difference in grafting method, liquid-phase grafting or vapor-phase grafting, produced no difference in those properties. The grafting was found to bring about a change in the molecular orientation of the original film.