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We investigated the molecular mechanisms responsible for the induction of apoptosis in mouse monocytic macrophage cell line J774A.1 stimulated by 7-ketocholesterol (7-KC). Cell apoptosis was detected by Annexin V-propidium iodide (PI) staining. The DNA-binding activity of nuclear factor kappa B (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that 7-KC-stimulation in J774A.1 cells activated NF-kappaB, which is involved in cell apoptosis, in a time- and dose-dependent manners. 7-KC was also found to increase the binding activity of NF-kappaB to specific DNA binding sites, a possible mechanism for the induction of the cell apoptosis. Moreover, these effects were partially inhibited by pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor. Taken together, 7-KC may be an important factor in atherosclerosis due to the ability of 7-KC to induce cell apoptosis, which is at least partially mediated through the activation of NF-kappaB.

Ciliary neurotrophic factor (CNTF) has been shown to decrease food intake in mouse models of obesity and to improve insulin sensitivity. It is well known that tight regulation of glucose metabolism is essential for successful gestational outcomes (e.g. fetal growth), and that abnormal insulin resistance is associated with preeclampsia (PE). To investigate the possibility that CNTF might be involved in the regulation of insulin resistance during pregnancy, circulating levels of CNTF were assessed in non-pregnant, normal pregnant, postpartum, and pregnant women with PE. Sera from healthy non-pregnant women (n10), pregnant women (n30:1st trimester;n10, 2nd trimester n10;3rd trimester;n10), postpartum women (n10), and patients with PE (n11) were studied with Western blotting. Circulating CNTF was detected by Western blotting, and the levels of CNTF in pregnant women were decreased as compared with those in non-pregnant women, and tended to decrease as pregnancy progressed. A significant decrease was found in PE as compared with normal pregnancy. Circulating CNTF might be associated with physiological and abnormal insulin resistance during pregnancy.

Our meticulous investigation of ePTFE graft breakage when a wire placed at the edge of an ePTFE graft was pulled, revealed that, depending on the breakage pattern, a break starts much earlier than the peak suture retention strength, which is the current international indicator for anastomotic-site break strength. Furthermore, the breakage patterns differ based on the thickness of the wire and the fiber direction of the ePTFE graft. Based on these findings, we advocate measuring the peak suture retention strength using 0.10-mm sutures and a standardized wire thickness in order to assess the anastomotic retention strength of ePTFE grafts.

Surfactant treatment in infants with respiratory distress syndrome (RDS) has decreased neonatal mortality. With the advent of this therapy, it has become important to predict accurately the fetal lung maturity of a fetus before delivery. We evaluated the stable microbubble test (SMT), surfactant protein-A (SP-A) and hepatocyte growth factor (HGF) in amniotic fluid as predicting markers for RDS. Of 55 amniotic fluid samples obtained by amniocentesis from women less than 37 weeks pregnant, the SMT values were as follows: sensitivity 76.5%, specificity 84.2%, positive predictive value 68.4%, negative predictive value 88.9% and overall accuracy 81.8%. For SP-A, the values were 88.2%, 65.8%, 53.6%, 92.6% and 72.7%, respectively. If we used both SMT and SP-A, we could diagnose with 100% accuracy that a case with measurements of SMT > or = 2 and SP-A > or = 420 ng/ml would not complicate with RDS (24/24). However, the RDS diagnostic accuracy of HGF does not equal to those of SMT and SP-A levels. We concluded that the rapidity, simplicity and reliability of SMT was very useful during 24-36 weeks of gestation as a bedside procedure to predict fetuses likely to develop RDS. We also noted the additive effect of SP-A in improving the accuracy of lung maturity diagnosis.

Changes in norepinephrine-induced transient contractions in Ca2+-deficient solution were investigated in the aortic smooth muscles of diabetic ALS (alloxan-induced diabetes susceptible) mice. The transient contractions in diabetic mice were significantly larger than those in normal mice. The longer incubation of the muscle preparations in Ca2+-deficient solution made the transient contractions smaller, probably due to the leakage and decrease in norepinephrine-releasable stored Ca2+. The rate of this reduction in contraction was slower in diabetic mice. These results suggest that the leakage of intracellular stored Ca2+ caused by extracellular Ca2+ deficiency is attenuated in diabetic mice, contributing to enhanced norepinephrine-induced transient contractions.

In considering the characteristics of the action of anxiolytic drugs and their mechanism in the brain, it may be necessary not only to study the behavioral pharmacology but also to perform brain site research. In the present study, the action of anxiolytic drugs was examined with respect to various behaviors that were induced by stimulating the brain areas related to emotions such as reward (pleasure) or aversion in rats. First, the low rate of response in lateral hypothalamic self-stimulation behavior was induced by schedules of low current brain stimulation, variable interval (VI) and differential reinforcement of low rate (DRL). Anxiolytic drugs such as benzodiazepines facilitated these low-rate responses. The drug susceptibility was highest in the low current stimulation, lower in the VI stimulation, and lowest in the DRL stimulation schedules. Furthermore, it was found by the auto-titration method in intracranial self-stimulation behavior that anxiolytic drugs decreased the threshold of stimulation reward. Second, it was recognized using the decremental lever pressing (DLP) paradigm that anxiolytic drugs increased the threshold of aversive stimulation of mesencephalic dorsal central gray (DCG), and this increasing effect of the drug was antagonized by GABA receptor blockers such as biccuculline. Finally, it was examined whether or not the conflict situation is established by combining brain stimulation reward and aversion, such as foot-shock or DCG stimulation. As a result, the conflict behavior was established by combining not only the brain stimulation reward and foot-shock aversion, but also the brain stimulation reward and DCG stimulation aversion. Further anxiolytic drugs exhibited anti-conflict action in both situations. The susceptibility of anxiolytic drugs was higher with respect to the conflict behavior induced by intracranial reward and aversion than to that induced by the conventional method based on milk reward and foot-shock aversion. These results suggest that behavioral methods using brain stimulation can examine the mechanisms of direct drug action at the brain stimulation site. Indeed, in the present brain stimulation behavioral study, anxiolytic drugs such as benzodiazepines increased the stimulation threshold in lateral hypothalamic self-stimulation and inhibited the DCG aversive stimulation, thus resulting in an anticonflict action of the drugs.

The purpose of this study was to search for chromosomal susceptibility loci for comitant strabismus. Genomic DNA was isolated from 10mL blood taken from each member of 30 nuclear families in which 2 or more siblings are affected by either esotropia or exotropia. A genome-wide search was performed with amplification by polymerase chain reaction of 400 markers in microsatellite regions with approximately 10 cM resolution. For each locus, non-parametric affected sib-pair analysis and non-parametric linkage analysis for multiple pedigrees (Genehunter software, http://linkage.rockefeller.edu/soft/) were used to calculate multipoint lod scores and non-parametric linkage (NPL) scores, respectively. In sib-pair analysis, lod scores showed basically flat lines with several peaks of 0.25 on all chromosomes. In non-parametric linkage analysis for multiple pedigrees, NPL scores showed one peak as high as 1.34 on chromosomes 1, 2, 4, 7, 10, 15, and 16, while 2 such peaks were found on chromosomes 3, 9, 11, 12, 18, and 20. Non-parametric linkage analysis for multiple pedigrees of 30 families with comitant strabismus suggested a number of chromosomal susceptibility loci. Our ongoing study involving a larger number of families will refine the accuracy of statistical analysis to pinpoint susceptibility loci for comitant strabismus.</P>

Photoelectric dyes absorb light and convert photon energy to electric potentials. To test whether these dyes could be used for retinal prostheses, a simple in vitro screening system was developed. Retinal neurons were cultured from the eyes of chick embryos at the 10-day embryonic stage, at which time no retinal photoreceptor cells have yet developed. Intracellular calcium elevation was observed with Fluo-4 in cultured retinal neurons before and after photoelectric dye was applied at varying concentrations to the culture medium. Five of 7 photoelectric dyes tested in this in vitro system induced intracellular calcium elevation in cultured chick retinal neurons. The intracellular calcium elevation generated by the 5 photoelectric dyes was blocked by extracellular calcium depletion in the case of all 5 dyes, and, except for one dye, by the presence of voltage-gated calcium channel blockers. The photoelectric dyes absorbed light under an inverted microscope and stimulated retinal neurons. This simple in vitro system allows the screening of photoelectric dyes which can be used for retinal prostheses.

The aim of this study was to reveal the clinical features of anterior urethral recurrence in patients with superficial bladder cancer, and to determine the appropriate treatment. Three hundred and three patients with superficial bladder cancer, who were newly diagnosed and initially treated conservatively in our hospital between 1965 and 1990, were followed for at least 5 years and their clinical outcomes were analyzed. Clinical factors, including anterior urethral recurrence, were evaluated statistically regarding tumor progression. Eight patients (2.6%) had anterior urethral recurrence following superficial bladder cancer. Twenty-four patients (7.9%) had tumor progression and 149 (49.2%) had tumor recurrence. In a multivariate analysis using a logistic model, anterior urethral recurrence was the most important factor, followed by histological grade. Four of 5 patients who were treated for anterior urethral recurrent tumors by transurethral resection showed progression and died of the cancer within one year. Two of the remaining three patients who underwent radical cysto-urethrectomy at the time of anterior urethral recurrence survived. Anterior urethral recurrence following superficial bladder cancer is a predictor for rapid subsequent malignant progression. Once there is anterior urethral recurrence, radical intensive therapy, including radical cysto-urethrectomy, should be carried out immediately.

High levels of soluble CD30 (sCD30) were detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA), indicating the involvement of CD30+ T cells in the pathogenesis. We investigated the induction of CD30 and its functions in CD4+T cells from patients with established RA (disease duration >_2 years). CD4+ T cells from both the peripheral blood (PB) and synovial tissue (ST) of RA patients expressed surface CD30 when stimulated with anti-CD3 antibody (Ab) and anti-CD28 Ab, but their CD30 induction was slower and weaker compared with PB CD4+ T cells of healthy controls (HC). Immunohistochemical analysis showed that only a small proportion of lymphocytes expressed CD30 in the ST (-1%). RA PB CD4+ T cells, after recovery from 6-day stimulation with anti-CD3 Ab and anti-CD28 Ab, showed in intracellular cytokine staining that CD30+ T cells could produce more interleukin-4 (IL-4) but less interferon-gamma. In the culture of RA PB CD4+ T Cells with anti-CD3 Ab and anti-CD28 Ab, blocking anti-CD30 Ab similarly inhibited the cell proliferation and activation of nuclear factor-kappaB on day 4 in RA and HC, but inhibited the apoptotic cell death on day 6 only in RA. These results indicate that despite high-level expression of sCD30, the anti-inflammatory activity of IL-4-producing CD30+ CD4+ T cells may be limited in the ST due to a poor induction of surface CD30 and a susceptibility to CD30-mediated cell death.

The purpose of this study was to elucidate the role of extracellular matrix components such as aggrecan, fibronectin, and laminin in the extraocular muscle of patients with strabismus. Resected tissues of the medial rectus muscle of 47 patients with intermittent exotropia obtained during recession-resection surgery were frozen under liquid nitrogen and pulverized by a Freezer/Mill to solubilize the tissue for enzyme immunoassay. The total amounts of aggrecan, fibronectin, and laminin in the resected tissue were correlated with clinical data of patients such as age, exodeviation, and refractive error. The amount of aggrecan decreased significantly with the advance of age (P < 0.0001, Spearman rank correlation test), while the amount of laminin or fibronectin had no correlation with age. Patients with basic type intermittent exotropia showed larger, although not significantly, amounts of aggrecan than those with convergence insufficiency type (P = 0.0538, Mann-Whitney U-test). The amount of aggrecan may be related to motor aspects of intermittent exotropia.

The effects of cepharanthin (Ce), glycyrrhizin (G), verapamil (V), and G plus V on induced thermotolerance in NIH3T3 cells were studied. Cells were heated with or without the drug at 45 degrees C for 20 min (the first heating), incubated at 37 degrees C for 12h (the incubation period), and heated again at 45 degrees C for 0-210 min (the second heating). G and V were added throughout the experiment, while Ce was added throughout the experiment or during only the first or second heating, or the incubation period. The cells were harvested after the second heating to evaluate cell survival. In control experiments without any drug, thermotolerance developed and reached the highest peak in the cells incubated for 12h at 37 degrees C. However, thermotolerance in the control cells was suppressed by incubating them at 0 degree C, but developed by subsequent incubation at 37 degrees C. This suggests that the acquisition of thermotolerance by the cells required metabolic processes during the incubation at 37 degrees C. When each drug was present throughout the experiment, only Ce or the combined use of G and V was effective in reducing thermotolerance. Thermotolerance was also suppressed in the presence of Ce during the second heating. These results indicate that Ce reduces thermotolerance by enhancing thermosensitivity rather than by inhibiting the development of thermotolerance.

Bronchial asthma was classified by the pathophysiology and by the mechanism of onset of the disease. Forty asthmatics who had serum IgE levels lower than 200 IU/ml were evaluated by two classification methods. 1. In asthma classified by a score based on clinical findings and examinations, the characteristics of the findings and examination results were compared among three asthma types, i.e., Ia. simple broncho-constriction type, Ib. bronchoconstriction+hypersecretion type, and II. bronchiolar obstruction type. Type Ib patients, in addition to manifesting hypersecretion, had a significantly higher proportion of eosinophils in the bronchoalveolar lavage (BAL) fluid compared to other asthma types. Significantly decreased values for ventilatory parameters and an increased proportion of BAL neutrophils were found in type II compared with other asthma types. 2. In a new classification by mechanism of onset, asthma was classified into three types according to the degree of participation of IgE-mediated reactions associated with specific IgE antibodies and serum levels of total IgE: asthma induced by definite IgE-mediated reaction (atopic asthma), possible IgE-mediated reactions (asthma), and asthma induced by non-IgE-mediated reaction (asthma syndrome).

Simultaneous radiohyperthermotherapy (SRH) is a combined hyperthermia-radiation therapy in which irradiation is given during heating. Mutual interference between the high energy radiotherapy system (Toshiba LMR-15A) and the 13.56 MHz capacitive heating system (Omron HEH-500C) was tested with phantom materials prior to a clinical trial with SRH. The energy and flatness of irradiation were not affected by the heating system within the range of clinical use. The high energy radiotherapy system did not affect the increase or distribution of temperature during simultaneous treatment. The results of this phantom study indicated that these apparatuses would not produce clinically significant mutual interference during SRH. A clinical trial was performed on a 57-year-old woman with postoperative recurrence of rectal cancer. This is the first reported clinical case treated with true SRH in which external irradiation was administered during mid capacitive heating. Twelve SRH treatments were performed on the recurrent lesion at a frequency of twice a week for six weeks using the apparatuses described above. There was a significant reduction in pain after treatment. The tumor marker carcinoembryonic antigen (CEA) level decreased after treatment. On CT images taken after treatment, the tumor site became a low density area which indicated necrosis. There were no side effects. These results suggest that further clinical study of SRH should be performed to clarify its advantages.

Southern blot hybridization was used to detect the rearrangement and amplification of five proto-oncogenes (bcl-2, bcl-1, c-myc, c-myb and c-Ha-ras) and one tumor suppressor gene (RB-1) in 55 Japanese patients with non-Hodgkin's lymphoma; 16 with T-cell lymphomas and 39 with B-cell lymphomas (7 follicular and 32 diffuse lymphomas). Genetic abnormalities of the proto-oncogenes were detected in 7 of the 55 (13%). Genetic abnormalities of bcl-2 plus other genes were detected in 5 of 7 cases of follicular lymphoma (71%), rearrangements of bcl-2 and c-myc, rearrangement of bcl-2 and amplification of c-myb. Genetic abnormalities were observed in only three cases of diffuse lymphoma. In each of 3 cases of B-cell lymphoma, one of the genes, blc-2 mbr, bcl-2 mcr and c-myc, was rearranged respectively. The incidence of genetic abnormalities in diffuse lymphomas (6.3%) was lower than that in follicular lymphomas. None of diffuse lymphomas had double oncogene abnormality. No abnormalities were found in RB-1, bcl-1, and Ha-ras. These findings suggest that follicular lymphomas are associated with some abnormalities of oncogenes not restricted to bcl-2 that facilitate growth which may be associated with their clinical features.

A cell line of human lung large cell carcinoma (LCC) was established directly from the metastatic skin tumor tissue. The clinical course of the patient who carried this carcinoma was peculiar; generalized lymphadenopathy, histologically resembling Hodgkin's disease, was found as the first clinical symptom. The lung tumor was not discovered until the time of autopsy. This cell line (KaMi) grew adherent to culture vessels with the population doubling time of 20.6h, formed colonies in soft agars with efficiency of 22.6%, and formed tumors in athymic nude mice. The authenticity of KaMi was confirmed by chromosomal analysis and isoenzyme patterns. KaMi cells bore a strong resemblance to the original tumor cells which were composed of small spindle cells, large polygonal cells, and multinucleated giant cells. Immunohistochemically, KaMi cells showed a weak tendency to differentiate to squamous cells, and these immunohistochemical reactivities were almost compatible to those of the original tumor cells, but ultrastructurally, KaMi cells were more immature than the original ones. Treatment with several reagents could not augment a differentiation of KaMi cells. Cytokeratin profiles showed a tendency of squamous cell differentiation. KaMi cells may aid in elucidating the pathogenesis and biology of LCC and its relationship to other lung tumors.

The results of our study may briefly be summarized as follows: 1) The irradiation with microrays (20∼30 watts) similar as 2,000 R and 5,000 R Gamma radiation did not substantially affect the activity of fibrinolysin (SK+SD). 2) By the irradiation method so far mentioned it has been demonstrated that the fibrinolytic activity of anticoagulant of the SK+SD preparation is preserved in all the clotting systems which we used. 3) Our findings indicate that it is possible to irradiate patients for therapeutical purpose with Radarmed (electromagneticrays) provided that there is produced some enhancing influence of the same blood clotting factors or systems. Together with earlier works in this field it appears that this method of the microirradiation could provide us with an important evidence on which we can base our further in vitro and in vivo radiohematologic studies; investigations with various preparations, types of radiation that are still underway9∼16.

2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of sulfhydryl reagents.