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Neural regulation of the motility between the haustra and taenia coli was studied in the isolated rabbit proximal colon. Four types of haustral and taenial preparations were used: the haustral strip without the taenia coli (type 1), the haustral strip including the taenia coli (type 2), the L-shaped (taenia-haustra) preparations for recording the haustral (circular) response to taenial stimulation (type 3) and the L-shaped (haustra-taenia) preparation for recording the taenial (longitudinal) response to haustral stimulation (type 4). Field electrical stimulation induced a contractile response in the haustra and taenia coli. Hexamethonium reduced the contraction in type 2, 3 and 4 preparations. The desensitization to serotonin reduced the response in type 2 and 3 preparations. After atropinization, the response in types 1 and 4 was reversed to relaxation, and the response in types 2 and 3 was reversed to relaxation followed by contraction which was reduced or abolished by indomethacin. The responses remaining after atropinization in all types of preparations were not affected by other blocking agents tested or desensitization to neuropeptides. Tetrodotoxin abolished all relaxation and contractile responses in all types of preparations. These results suggest that the indirect contractile response to field stimulation is induced mainly via cholinergic and serotonergic neurons, and that the relaxation is mainly mediated by nonadrenergic noncholinergic neurons. The late haustral contractions after atropine may be caused by endogenous prostaglandin.

We examined the effect of fetal calf serum (FCS) on meiotic division, subsequent fertilization, and first cleavage to the 2-cell stage of rat oocytes during in vitro maturation. FCS had no effect on the nuclear progression from dictiate to metaphase of the second maturation in vitro and, FCS had no effect on the first cleavage to the 2-cell stage of fertilized oocytes. However, FCS efficiently increased penetration rate of oocytes and shortened the time required for dissolution of the zona pellucida by alpha-chymotrypsin. These results showed that FCS did not affect cytoplasmic maturation necessary for oocytes to develop to the 2-cell stages. We found that FCS only affects the zona pellucida and does not affect the nucleus or cytoplasm of rat oocytes. FCS may prevent hardening of the zona pellucida.

Ki-67 is a commercially available mouse monoclonal antibody (MoAb), which reacts with a nucleolar antigen (the Ki-67 antigen) expressed in proliferating eukaryotic cells. The author examined the precise localization of the Ki-67 antigen in C-6 cells using immunohistochemical and immunoelectron microscopic methods and estimated the proliferative activity of human brain tumors in situ. Positive nucleoplasmic reactions (early G1 phase) and nucleolar staining (late G1 phase) were observed. The cells showed very weak positive reactions in only one or two nucleoli (S phase) and multiple spicule reactions in the nucleoplasm (G2 phase). During the mitotic phase, the Ki-67 antigen was stained on the surfaces of all chromosomes and finely dispersed in the cytoplasm. By immunoelectron microscopic study, positive reactions were observed on the granular and dense fibrillar components. Therefore, the Ki-67 antigen seems to participate in the processing and assembly of preribosomal particles. In human brain tumors, the Ki-67 score (positive cells/total neoplastic cells), ranging 0 to 36.7%, correlated well with the histopathological grade of malignancy of the tumor. These findings suggest that immunohistochemical staining with the MoAb Ki-67 can be used as a convenient procedure for the simple evaluation of the proliferative activity of brain tumors.

Carbohydrate metabolism of rats with obstructive jaundice caused by bile duct ligation was studied by intravenous glucose tolerance test (IVGTT) and by liver perfusion. The altered levels of carbohydrate-metabolizing enzyme were examined in relation to the glucose metabolism of the cholestatic rats. In the IVGTT, the rate of fractional glucose removal was increased with increases in plasma insulin and glucagon and with a decrease in non-esterified fatty acid. In liver perfusion, neither the glucose uptake nor insulin extraction by the whole liver of icteric rats was different from the control. The increased rate of glucose removal in IVGTT may be due to enhanced glucose utilization by peripheral tissues resulting from hypersecretion of insulin. In liver perfusate supplemented with glucose, a decrease in the glucose uptake per unit liver weight was observed in relation to the lowered glucokinase activity. Formation of glycogen from glucose and of glucose from lactate was also impaired, indicating inhibition of the gluconeogenic system or relative hyperfunction of the glycolytic system, which may further contribute to the reduction in glycogen content. These metabolic disorders correlated well with the changes in activities of key carbohydrate-metabolizing enzymes, which showed a characteristic pattern consistent with the loss of differentiated hepatic functions. Uptake of glucose and its conversion to glycogen were reduced in the cholestatic liver in close association with altered activities of some of related enzymes. However, due to increased utilization by the peripheral tissues, the total amount of glucose utilized in the whole rat was not reduced.

To prevent the development of hepatic metastases after surgery for colorectal cancer, it is important to inhibit the growth of any micrometastases which occur during the operation. We used a hepatic metastasis model in mice to investigate the effects of combination therapy with natural human tumor necrosis factor-alpha (nHuTNF-alpha) and natural murine interferon-alpha/beta (nMuIFN-alpha/beta). Decreased formation of hepatic metastases by murine colon-26 carcinoma was recognized following a single injection of nHuTNF-alpha, nMuIFN-alpha/beta, or both. These inhibitory effects were synergistic. NK activity was also measured, because notaral lerller cells not only have an anti-tumor effect but are also a representative of the host immune system. Both nHuTNF-alpha and nMuIFN-alpha/beta were able to activate NK cells, and the combination of the cytokines more significantly augmented NK activity. The in vivo elevation of NK activity induced by nHuTNF-alpha, nMuIFN-alpha/beta, or their combination may be one of the mechanisms of their antiproliferative effect on experimental hepatic metastases of murine colon-26 carcinoma.

In 15 patients with Japanese cedar pollinosis and 10 healthy control subjects, levels of major basic protein (MBP), eosinophil cationic protein (ECP), and arylsulfatase B (As) in the nasal secretions were examined before and after challenge with Japanese cedar pollen extract. The MBP and ECP levels in the patients were significantly higher 30 min after challenge than those before challenge (P < 0.005). MBP and ECP levels after challenge were significantly higher in the nasal secretions of patients than in the controls (MBP: P < 0.01, ECP: P < 0.05). The level of As after challenge was significantly higher in the nasal secretions of patients than in the controls. These results suggest that eosinophils activate or modify the immediate, nasal allergic reaction and have a role in regulating immunological responses.

To elucidate the effect of the arginine vasopressin (AVP) system in vivo, especially V1 and V2 activity, on blood pressure, we measured the acute changes in blood pressure and heart rate after AVP, OPC-21,268 (a V1 receptor antagonist), and OPC-31,260 (a V2 receptor antagonist) were injected intravenously in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at the age of 15 weeks. Compared with the control period, single injection of AVP 5 ng/kg significantly increased systolic blood pressure in WKY rats without a concomitant increase in heart rate, but there was no significant increase in blood pressure in SHR. In contrast, single injection of either OPC-21,268 3 mg/kg or OPC-31,260 3 mg/kg did not affect blood pressure or heart rate in either SHR or WKY rats. Injection of AVP after the administration of OPC-31,260 induced a greater increase in blood pressure in SHR than in WKY rats, whereas injection of AVP after the administration of OPC-21,268 did not induce any clear increase in blood pressure in SHR or WKY rats. These results suggest that SHR have enhanced pressor activity mediated by V1 receptors and that this increase may be due to an increase in their number. In conclusion, enhancement of V1 activity may contribute to the development of high blood pressure in SHR.

In the fields of psychiatry and neurology, the dopaminergic system is one of the most important neurotransmitter systems in the brain. Whereas pharmacological and biochemical studies had initially indicated two subclasses of dopamine receptors (DA-R), recent progress in molecular biology techniques has led to the identification of five distinct genes of DA-Rs (D1-R-D5-R) and splice variants. The gene products are classified into the D1-R family (D1-R and D5-R) and D2-R family (D2-R, D3-R and D4-R) based on their structure and pharmacological features. This review summarizes the structure, localization, function and pharmacology of DA-R subtypes on the basis of knowledge obtained during the past few years. The genes encoding the D1-R family have no intron and the D2-R family genes have introns. The distributions of mRNAs encoding these five DA-R subtypes in the brain were different from their respective receptors. The localization of DA-R subtypes to particular brain regions and specific pharmacological profiles of DA-R subtypes allow new insights to be made into the mechanism of action of DA in the control of psychiatric and motor functions. The availability of detailed information about DA-R subtypes will not only clarify their roles in the brain, but will probably also lead to the development of new therapeutic drugs with more specific actions.

The establishment of a model system of neoplastic transformation of normal human cells has been attempted with a chemical carcinogen, 4-nitroquinoline 1-oxide (4NQO). In the course of these experiments, it was noticed that immortalization of human cells is a multi-step process involving several mutational genetic events. Thus, chromosomal changes which occurred during the process of immortalization of human fibroblasts were examined. To accomplish immortalization, fibroblasts obtained from an embryo were repeatedly treated with 10_-6M4NQO from primary culture to passage 51 (59 treatments in total). Before immortalization, some chromosomes (especially, chromosomes 2, 6, 8, 10, 11, 12, 15, 19, and 20), were lost at a relatively high frequency. After immortalization, the chromosomes distributed so broadly in the triploid to hypotetraploid region without a distinct modal number or without marker chromosomes that it was difficult to identify the specific chromosomes related to the immortalization of human cells. No specific structural chromosomal changes were detected. Although the significance of such chromosome changes in relation to immortalization is not clear, the loss of some specific chromosomes suggests that genes which are involved in cellular aging and which suppress immortalization may have been lost in the immortalization process.

Tissue PIVKA-II was examined in 32 hepatocellular carcinomas and 2 metastatic liver tumors using indirect immunofluorescence, and the results were compared with the size, histological grading and serum PIVKA-II level. The specificity of this method was confirmed by the disappearance of reactivity in PLC/PRF/5 cells after the addition of vitamin K to the culture medium. Positive PIVKA-II staining was observed as a clustered or a single cell pattern only in the HCC nodules, but not in the surrounding cirrhotic tissue. PIVKA-II staining was observed in all HCC groups regardless of histological grade. There was no relationship between PIVKA-II staining and the size of HCC. PIVKA-II was detected immunohistochemically even in small HCC of patients whose plasma PIVKA-II levels were below the detection limit. These results suggest that PIVKA-II production is a specific phenotype of HCC regardless of its histological grading and demonstrate that this immunofluorescent PIVKA-II staining is more sensitive and useful than plasma PIVKA-II assay for the diagnosis of HCC.

In single treatment study, ethanol was administered intraperitoneally to ICR mice (about 34 g) in the amounts of 1.0, 2.0, 3.0 or 4.0 g/kg body weight. The 3,4-dihydroxyphenylacetic acid (DOPAC) + homovanillic acid (HVA) concentration in the striatum was elevated with 3.0 and 4.0 g/kg of ethanol. In the hypothalamus, the DOPAC, HVA and 5-hydroxyindoleacetic acid concentrations were increased after injection of 3.0 and 4.0 g/kg of ethanol. Furthermore, the acetylcholine (ACh) and gamma-aminobutyric acid (GABA) concentrations were also increased following the injection of 1.0, 2.0, 3.0 and 4.0 g/kg. To study the effects of repeated administration, mice were injected intraperitoneally with 1.0 or 2.0 g/kg of ethanol once daily for 7 days. The DOPAC + HVA level in the striatum was elevated after injection of 1.0 and 2.0 g/kg of ethanol. The GABA and ACh concentrations in the hypothalamus were decreased after repeated injections of ethanol. These results suggest that ethanol significantly alters the utilization of dopamine, ACh and GABA in the hypothalamus. This may partially explain why ethanol has such profound effects on emotional behavior and mood.

Stress distribution in the first carpometacarpal joint was analyzed in 49 cadaveric hands using the finite element method to clarify the pathogenesis of osteoarthritis in the joint. The results of the finite element method analysis were compared with those of the contact pressure distribution in the first carpometacarpal joint of cadaveric specimens using pressure-sensitive film, and with the simple roentgenographical and microradiographical manifestations of spur formation, and with histological findings of osteoarthritis to verify the accuracy of the models of computer simulation models. The comparison of these results showed that osteoarthritic changes of the first carpometacarpal joint were found in areas where stress was concentrated during movement of the joint. The saddle shape of this joint is essentially well-designed for the dispersion of normal stress, however minimal displacement due to instability could easily induce osteoarthritis. Furthermore the shallow trapezial configuration may contribute to the high incidence of osteoarthritis changes. The finite element method helped clarify the relationship between stress patterns and osteoarthritis response.

The effects of intraperitoneal administration of 2-(4-carboxy-D-gluco-tetrahydroxybutyl)thiazolidine-4-carboxylic acid (CGUA), a cysteine derivative conjugated with glucuronic acid, on total glutathione and total cysteine contents in rat tissues were investigated. Total glutathione (GSH and GSSG) and total cysteine (cysteine and cystine) were determined by a new method consisting of preparation of S-carboxymethylglutathione (CMSG) and S-carboxymethylcysteine (CMC), respectively, and subsequent analyses with an amino acid analyzer. CGUA was determined by a coloration method employing an acidic ninhydrin reagent. Total cysteine contents in liver, kidney and plasma rapidly increased to 2.3, 2.7 and 6.5 times the levels of the controls, respectively, after CGUA administration at a dose of 5 mmol/kg of body weight. Total glutathione content did not change significantly in the liver or blood except for the kidney with a significant increase during the first 1-h period after administration. CGUA content increased markedly in these tissues, especially in the kidney, and 30% of administered CGUA was excreted in urine within 2h. These results indicate that CGUA is converted into cysteine in vivo, suggesting the usefulness of this compound for protection of the kidney and the liver.

Left ventricular assist device (LVAD) was utilized for the treatment of postcardiotomy heart failure in two patients with Marfan's syndrome. Patient 1 (a 22-year-old) with annuloaortic ectasia (AAE) and DeBakey type II dissection had been supported by LVAD for 87h after composite graft replacement of the ascending aorta and aortic valve. Patient 2 (a 52-year-old) with AAE and DeBakey type I dissection had been supported by LVAD for 91 h after aortic valve replacement. During the assist, both patients complicated bleeding from the fragile left atria near the sites of cannulation. Patient 1 died of multiple organ failure on the 62nd postoperative day, but patient 2 returned to work after surgery.

"Free" iron, a potentially radical-generating low mass iron, and not found in normal human blood, was increased in the serum of blood-transfused thalassemia major patients seen in the Yangon General Hospital, Yangon, Myanmar (Burma). The low mass iron was detected by the bleomycin assay. Fifty-one blood samples were analyzed (from 28 males and 23 females). High "free" iron was detected in 47 sera samples from thalassemia patients. Serum ferritin, which reflects the body store iron, was higher than the normal range (10-200 ng/ml) in 49 patients. On the other hand, serum iron of 39 sera samples fell within the normal range (50-150 micrograms/dl). Four were less than 50 micrograms/dl and eight were more than 150 micrograms/dl. Almost all the patients' sera of normal or higher serum iron level contained "free" iron. Thus, almost all the sera from thalassemic patients from Myanmar contain bleomycin-detectable iron, even when serum iron is within the normal range. In developing countries where undernutrition is prevalent (serum albumin in these patients was 3.6 +/- 0.4 g/dl, P < 0.0001 vs. control value of 4.0 - 4.8 g/dl), normal serum iron does not preclude the presence of free iron in the serum.

To study the virological and serological characteristics of asymptomatic hepatitis C virus (HCV) carriers, 165 blood donors positive for antibody against HCV proteins by the second generation assay, were analyzed for their clinical backgrounds, serological reactivity against antigens derived from HCV by recombinant immunoblot assay, and the amount and genotype of HCV by the polymerase chain reaction. Compared with blood donors having abnormal levels of alanine aminotransferase (ALT), sera from the donors with normal levels of ALT reacted less frequently against NS4 antigens (anti-5-1-1: 34.4% vs. 54.5%, P = 0.0609; anti-c100-3: 34.4% vs. 56.1%, P < 0.05). Also the positivity for antibodies against these antigens were more frequent in sera from donors with genotype 1b HCV-RNA than other genotypes (anti-5-1-1: 61.0% vs. 23.5%, P < 0.01; anti-c 100-3: 61.0% vs. 26.5%, P < 0.01). The prevalence of each genotype in blood donors with normal ALT levels was different from that in patients with advanced liver disease (P < 0.05), genotype 1b being less and genotype 2a being more frequent. The number of HCV-RNA copies/0.5 ml in donors with normal ALT was 10(7.9 +/- 1.0) (n = 27) and that in patients with chronic liver disease was 10(7.4 +/- 0.8) (n = 116), the difference being statistically significant (P < 0.05). In conclusion, the results of this study suggest that asymptomatic blood donors carrying HCV have the serological and virological characteristics different from the patients with advanced liver disease.

To find an effective way to handle wheelchairs, 3-dimensional floor reactions of the hand and angular deviation of the elbow and wrist joints during push-up motion were studied in 10 healthy men. The push-up was carried out using 3 hand positions (fist, finger and palm) and a push-up device. In all hand positions, anteroposterior force (Fx) and the mediolateral force (Fy) appeared after the vertical force (Fz). The end point of Fx and Fy was observed before that of Fz. Among the 4 different hand positions, Fx and Fy appeared first in the palm, followed by the finger and fist positions, and lastly in the push-up devices. The results indicate that the more unstable pushing-up the body is, the earlier and longer Fx and Fy are. Thus, Fx and Fy are considered to be good indicators of body balance during the push-up motion. The elbow joint showed a hyperextended position only when using the palm position in the maintenance phase. The wrist joint showed palmar flexion only when using the fist position.

A persistent problem in orthotopic liver transplantation is primary nonfunction (PNF) of the hepatic allograft. In an attempt to reduce the incidence of graft failure, the feasibility of pretransplant assessment of graft viability was investigated by 31P nuclear magnetic resonance (NMR) spectroscopy. The level of adenosine triphosphate (ATP) was measured as an indicator of liver function by 31P NMR spectroscopy after a 30 min normothermic reperfusion following cold-storage in University of Wisconsin (UW) solution. The mean +/- SD beta-ATP/Pi ratio after preservation for 0, 12, 24 or 48 h was 1.40 +/- 0.34, 0.85 +/- 0.27, 0.64 +/- 0.14 and 0.38 +/- 0.09, respectively. Significance was observed between 12h and 24h and between 12h and 48h of preservation. These results correlated well with the morphological changes in endothelial cells and sinusoidal lining cells examined by transmission electron microscopy. It is suggested strongly that microcirculatory disturbances due to endothelial cell injury impairs the recovery of ATP levels after reperfusion, and that ATP determination by 31P NMR spectroscopy, as a non-invasive modality, may help in the prediction of PNF after liver transplantation.