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The effects of laminin (LAM) and collagen type I (C-I) on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines were investigated. C-I was superior to LAM in supporting the attachment of the cells, especially of HuH-6, to plastic surfaces. No effect of LAM and C-I on cellular morphology was recognizable by phase contrast microscopy. By scanning electron microscopy (SEM), much more microvilli were found on the cell surface of HuH-6 on LAM substrate than on C-I substrate. In HuH-7 cells, however, these microvilli were rarely found on either LAM substrate or C-I substrate. The gel profile of the proteins secreted by HuH-6 and HuH-7 cells was not affected by the culture substrate except for the major band, though the amount of alpha-fetoprotein (AFP) secreted was larger when the cells were cultured on LAM substrate than on C-I substrate. These results indicate that the ability of LAM or C-I to enhance attachment is different from that to enhance AFP production or microvilli expression in HuH-6 cells and probably in HuH-7 cells.

A new method for staining sialoglycoproteins in polyacrylamide gel after disc electrophoresis is described. The method utilizes the reaction of sialic acids with an acidic ninhydrin reagent which yields a stable color with an absorption maximum at 470 nm. After electrophoresis, the polyacrylamide gel is placed in a test tube and heated with 5 ml of the acidic ninhydrin reagent for 10 min in a boiling water bath. Sialoglycoproteins are detected as brown bands. No additional procedure such as destaining is necessary. When 20 micrograms fetuin, a sialoglycoprotein, per gel is applied, the band remains visible for at least 2 h. Stained gel can be scanned with a gel scanner at 470 nm. When the stained gel was dried on a sheet of polypropylene filter, the color was stable for at least one month. The present method is superior to the method using Stains-all (3,3'-diethyl-9-methyl-4,5,4',5'-dibenzothiacarbocyanine) in specificity and simplicity for the detection of sialoglycoproteins.

The concentration of lipoperoxides in maternal blood increases as gestation progresses. The concentration in pregnant women at 40 weeks gestation is 1.6 times higher than in nonpregnant women. The concentration in the cord blood, however, is 70% lower than that in maternal blood. To study the role of placental tissue in the difference in the lipoperoxide concentration between the cord blood and maternal blood, we investigated the lipoperoxide concentration, antioxidant activities and in vitro lipoperoxide formation in placental tissue during pregnancy. The lipoperoxide concentration was 50% lower in placental tissue of 40 weeks gestation than in tissue of 5-11 weeks gestation. Catalase and superoxide dismutase activities in placental tissues increased as gestation progressed, while glutathione peroxidase activity and alpha-tocopherol concentration did not change significantly during the gestational period. The in vitro formation of lipoperoxides in placental tissue decreased as gestation progressed. These results show that placental tissue suppresses lipoperoxide formation in the late gestational age, lowers the concentration of lipoperoxides in the blood and protects the fetus against oxygen toxicity.

We made posterior hypothalamic knife cuts in rats to transect the fibers of the medial forebrain bundle (MFB) at the level of the mammillary body. The role of the MFB in the baroreflex and hemorrhage-induced hormonal responses was then examined in the unanesthetized, freely moving condition. The slopes for the relationship between changes in pulse interval and mean arterial pressure (MAP) in the posterior-cut group were significantly steeper than those in the sham-cut group both when there were phenylephrine-induced increases in MAP (1.13 +/- 0.07 vs 0.86 +/- 0.10 msec/mmHg) and nitroprusside-induced decreases in MAP (1.16 +/- 0.10 vs 0.77 +/- 0.05 msec/mmHg). This result indicates that posterior cuts elevated baroreflex sensitivity when MAP was increased or decreased. The resting MAP was not changed, but the resting heart rate (HR) was lowered by the posterior cuts. Furthermore, the posterior cuts augmented hypotensive hemorrhage-induced bradycardia. Hypotensive hemorrhage (16-17 ml/kg) caused elevation of the plasma catecholamine, ACTH and vasopressin (AVP) levels, but the posterior cuts attenuated these hormonal responses. These results indicate that the fibers in the MFB have a tonic inhibitory effect on the baroreflex in the resting condition, and play a stimulatory role in hemorrhage-induced catecholamine, ACTH and AVP responses.

Meth A-fibrosarcoma bearing BALB/c mice were subjected to selected splenic irradiation (2.0-4.0 Gy) on days 7 and 14 of tumor growth. Tumor growth was recorded by serial measurement. Irradiation given on day 7 caused regression of tumor, but irradiation given on day 14 did not show tumor regression. Antitumor activity in the Winn assay was detected in spleen cells 3 days after irradiation, but was not detected 7 days after. The cell surface phenotypes were analyzed on days 3, 7 and 14 of splenic irradiation using monoclonal antibodies (anti-Thy1.2 antibody, anti-Lyt1 antibody, anti-Lyt2 antibody, anti-L3T4 antibody) by flow cytometry. Thy 1.2, Lyt1, and L3T4 cells were increased on day 3 of splenic irradiation, but were not on days 7 and 14. Lyt2-cells did not show increase on days 3, 7 and 14. It was possibly suggested that selected splenic irradiation induced tumor regression was caused by the ability of irradiation to preferentially eliminate suppressor T cells, thereby allowing effector T-cells to become relatively dominant.

The effects of uridine(UR) on the cell-killing activity of cytosine arabinoside(ara-C) against human leukemic cells, MOLT-4, and on ara-C accumulation in cells were studied. The 50% lethal dose(LD50) of ara-C as determined by clonogenic assay was decreased to 5.0 x 10(-8) mol from 9.0 x 10(-7) mol after 3 days exposure to 10(-3) mol of UR. The accumulation of 3H-ara-C at 24 and 48 h was significantly increased in culture medium containing 10(-8) mol of 3H-ara-C and 10(-3) mol of UR (5,129 +/- 123.5 vs 2,554 +/- 115.5 cpm/10(5) cells at 24 h, p less than 0.01, and 5,772 +/- 123.2 vs 1,372 +/- 51.8 cpm/10(5) cells at 48 h, p less than 0.01). It is noteworthy that cell-killing activity of ara-C against human leukemic cells was enhanced by the combination with a nucleoside(UR), but not with antileukemic agents.

The purpose of this study was to evaluate the role of arachidonic acid metabolites in the reimplantation response after lung transplantation in mongrel dogs. The left lung was used and two groups were studied. Group I underwent hilar stripping, while Group II underwent hilar stripping plus warm ischemia for 60 min., achieved by clamping the left pulmonary artery and veins. We measured the lung wet to dry weight ratio (W/D ratio), total pulmonary vascular resistance (TPVR), and blood and bronchoalveolar lavage fluid (BALF) levels of leukotriene B4 and C4 (LTB4,C4) and thromboxane B2 (TXB2). These parameters were measured periodically for 7 days after reperfusion. In group II, the W/D ratio and TPVR were significantly increased in comparison with Group I. The blood LTC4 level was elevated immediately after reperfusion, and BALF level of LTC4 also rose subsequently. These levels changed concomitantly with the W/D ratio. The above results suggest that arachidonic acid metabolism plays an important role in the reimplantation response, especially in pulmonary edema.

Twenty-four patients with rheumatoid arthritis (RA) and 20 normal controls were examined for the ability of their peripheral blood B cells to produce interleukin 1 (IL-1) with or without lipopolysaccharide (LPS). B cells were purified from peripheral blood by negative selection methods (i.e., removal of adherent cells and sheep red blood cell rosette-forming cells, followed by treatment with monoclonal antibodies (OKT3 and OKM1) and complement). The amount of IL-1 in B cell culture supernatants (SN) was measured by thymocyte and fibroblast proliferation assays and an enzyme-linked immunosorbent assay for IL-1 alpha and beta. As a group, cultured B cells from patients with RA, both spontaneously and when stimulated with LPS, produced higher levels of IL-1 than those from normal controls. IL-1 production by RA B cells with LPS had a weak but positive correlation with disease activity. Moreover, RA B cell culture SN with elevated levels of IL-1 had a synergistic effect on the growth of anti-human IgM (anti-mu) stimulated B cells. In separate experiments, the growth of RA B cells was significantly promoted by IL-1 beta both with and without anti-mu stimulation. These results suggest that B cell-derived IL-1 may be involved in the B cell clonal expansion of RA through its own activity as a B cell stimulatory factor.

Pulmonary function tests were performed on 234 healthy non-smoking young subjects (189 males and 45 females free from respiratory and allergic symptoms). Maximal expiratory flow-volume (MEFV) curves were visually classified into five MEFV types: Type A, convex or straight flow changes; types B, C, and D, concave-convex-concave flow changes; and type E, sudden flow-fall and accompanying decreased flow rates at lower lung volumes. The reproducibility of MEFV patterns were shown by one way analysis of variance (ANOVA) of MEFV data obtained from 4 groups each consisting of 3-4 males and representing different MEFV types. Distribution of MEFV types was different between males and females; the rate of type A was higher in females than in males and those of types B and E were higher in males than in females. When analyzed in terms of three fractional flow rates, Fr-75, Fr-50, and Fr-25, these values could also be classified into 5 types similarly to the visual MEFV type analysis. It is concluded that MEFV type analysis is useful in assessing health conditions.

We demonstrated the ultrastructure of rat glomerular basement membrane (GBM) by ultra-high resolution scanning electron microscopy. GBM prepared by sonication methods and conductive-staining could be observed without metal coating at magnifications as high as 400,000 times. The GBM showed an irregular meshwork structure composed of various strands and pores. The width of the strands ranged from 6 to 15 nm, and the diameter of pores ranged from 6 to 50 nm. The present study confirmed our molecular sieve theory of the basement membrane.

The cytotoxic effects of ferric nitrilotriacetate (Fe-NTA) have been considered to be caused by free radicals produced by the drug. The present study was carried out to determine whether or not cytotoxic effects of Fe-NTA on cell growth and lipoperoxide formation of Chinese hamster cells were reduced by antioxidants. Using a spin trapping technique, we found that hydroxyl radical formation in the cells increased in the presence of Fe-NTA. Antioxidants, with the exception of superoxide dismutase, slightly inhibited production of the hydroxyl radical. Mannitol significantly reduced lipoperoxide formation, but other antioxidants did not. However, the growth inhibitory effects of Fe-NTA were not attenuated by these antioxidants. These results indicated that the cytotoxic effects of Fe-NTA may be mostly due to unknown factors other than oxygen free radicals.

A 56-year-old man was admitted to our department with a chief complaint of lower extremity dysesthesia. He described a dull numbness below the ankle and a dull pain in the nates for the past two years. Although the numbness extended to the thigh, he did not notice any muscular weakness or atrophy. Neurological examination revealed weakness and atrophy in the face, tongue and the proximal portions of all four extremities. Deep tendon reflexes were decreased. A moderate loss of vibratory sensation was noted below the knees. Electromyography showed neurogenic changes. Muscle biopsy revealed both myogenic and neurogenic changes. Sural nerve biopsy revealed a mild reduction of myelinated fibers, particularly the large-diameter fibers. Based on these findings, a diagnosis of bulbospinal muscular atrophy (BSMA) was made. In recent years, there have been some case reports of BSMA with sensory disturbances, or merely with subclinical manifestations of a sensory disturbance. This case is included in the same category as those reports, but it is interesting to note that the sensory disturbance in the lower extremities occurred as the chief complaint of the disease.

We report a case of a non-Hodgkin's lymphoma (NHL) patient treated successfully with combination chemotherapy during pregnancy who delivered a full-term baby. A 29 year-old patient with cervical and inguinal lymphadenopathy in the 27th week of gestation was referred to our hospital. The diagnosis of lymph node biopsy was NHL (diffuse, large cell type with B-cell phenotype). Three courses of CHOP regimen (adriamycin, cyclophosphamide, vincristine and prednisolone) were given before delivery. The patient has been in complete remission for three years and her baby has been in normal development. Our case supports previous reports that chemotherapy in the third trimester may be given safely on NHL patients.

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.