Human papillomaviruses associated with cervical intraepithelial neoplasia. Great diversity and distinct distribution in low- and high-grade lesions. All together, 30 genital human papillomavirus (HPV) types have been characterized so far. To evaluate the importance of HPV diversity in associated cervical diseases, we analyzed 188 biopsy specimens obtained from patients with a recent diagnosis of cervical HPV infection or intraepithelial neoplasia (CIN). Of these 188 specimens, 116 were classified as low-grade CIN (48 cases), high-grade CIN (53 cases), condylomata acuminata (10 cases), flat condylomas (five cases). Seventy-two specimens were considered nondiagnostic. Using probes specific for 18 genital HPV types, HPV DNA sequences were detected by Southern blot hybridization in 100 lesions and 21 nondiagnostic specimens. When further analyzed by the polymerase chain reaction, eight HPV-negative biopsy specimens, four CIN, and four nondiagnostic specimens were positive. Of the 129 positive biopsy specimens, 92 contained at least one of 18 known HPV types and 37 HPV that have not yet been identified. Nine specimens had more than one type. Thirteen HPV types were identified in CIN. The detection rate of HPV 16 increased from 21% in low-grade CIN to 57% in high-grade CIN. HPV 18 was detected in only 3% of CIN; HPV 31, 33, and 35 were found in 8%. HPV 30, 39, 45, 51, 52, 56, 58, and 61 were detected in 44% of low-grade CIN but in only 8% of high-grade CIN. Unidentified HPV were detected in about 25% of low-grade and high-grade CIN. Fifty-seven CIN positive for at least one HPV type were further analyzed by in situ hybridization. Thirty-five (65%) biopsy specimens were positive, including 21 of 24 low-grade CIN and 14 of 33 high-grade CIN. Ten of the 13 previously identified HPV types were detected. Thus, CIN represents an heterogeneous disease from a virologic viewpoint. This fact could explain their variable clinical evolution.


Fibrosarcoma-malignant fibrous histiocytoma of the breast. A clinicopathological study of 32 cases. We report the clinical and pathologic features of 32 sarcomas of the breast with features spanning the spectrum of fibrosarcomas-malignant fibrous histiocytomas. Neoplasms were categorized as high- or low-grade lesions depending on a combination of the degrees of atypia and mitotic activity. The majority of high-grade lesions had marked (3+) nuclear atypia and at least five mitotic figures per 10 hpf. High-grade lesions with moderate (2+) nuclear atypia had a mitotic activity of six or more mitotic figures per 10 hpf. All low-grade lesions had five or fewer mitotic figures per 10 hpf, and none had a score of the nuclear grade times mitotic figures of more than 10. The average mitotic activity in low-grade lesions was two mitotic figures per 10 hpf; the high-grade lesions had 12 mitotic figures per 10 hpf. Sixty-nine percent of the low-grade fibrosarcomas-malignant fibrous histiocytomas showed mild (1+) cytologic atypia, and 69% of the high-grade lesions showed severe (3+) cytologic atypia. The herringbone pattern was associated with a more favorable prognosis than the malignant fibrous histiocytoma pattern. Compared to the high-grade lesions, low-grade fibrosarcomas-malignant fibrous histiocytomas were slow-growing, produced fewer recurrences, and did not metastasize. Of the 16 women with low-grade lesions, all were free of tumor at last contact, despite recurrence in more than half of the patients. In contrast, 31% of the patients with high-grade lesions died of tumor, and 13% were alive with disease. Twenty-five percent of women with high-grade lesions developed distant metastases.


Comparison of extracellular matrix antigens in subtypes of bronchioloalveolar carcinoma and conventional pulmonary adenocarcinoma. An immunohistochemical study. In contrast to the conventional pulmonary adenocarcinomas (CPAs), bronchioloalveolar carcinoma (BAC) grows predominantly by spreading along the existing alveolar septal framework. Within the BAC category, three subtypes have been identified: mucinous, nonmucinous, and sclerosing BAC. Of these, mucinous and sclerosing BACs have worse prognoses compared with nonmucinous BAC. However, the manifestation of aggressive behavior is different between the mucinous and sclerosing types of BACs. Multifocality is often produced by aerogenous spread, especially in the case of mucinous BACs. To study the differences between the BAC subtypes and the conventional pulmonary adenocarcinomas, we employed a battery of immunohistochemical stains marking the extracellular matrix architecture (laminin, collagen IV, fibronectin, and collagen III), a degradative enzyme against a basement membrane component (anti-type IV collagenase) and cellular receptors for laminin and collagen IV (alpha 2 integrin) on 16 BACs (5 mucinous, 5 nonmucinous, and 6 sclerosing) and 30 CPAs. The mucinous and nonmucinous BACs demonstrated neoplastic epithelial cells growing along a continuous basement membrane. A similar growth pattern with intact basement membrane was noted in the periphery of sclerosing BACs. However, in contrast to mucinous and nonmucinous BACs, all cases of sclerosing BACs showed disruption or complete absence of basement membrane components (laminin and collagen IV) around the embedded glands located centrally in the sclerotic fibrous stroma, as was seen in the basement membrane analysis of conventional adenocarcinomas. Furthermore, increased type IV collagenase activity was seen in the small centrally located embedded glands in comparison to the peripheral glands. These architectural alterations of basement membrane disruption and phenotypic expression of degradative activity may be a reflection of the invasive behavior of the sclerosing BACs and their tendency to produce lymph node metastasis. Although the mucinous BACs did not show evidence of basement membrane disruption, there was a marked increase in their levels of type IV collagenase expression along with consistently low levels of alpha 2 integrin receptor (laminin and collagen IV receptor) expression. These findings may be related to the ability of the mucinous BACs to detach from the underlying basement membrane and spread aerogenously, and is to be contrasted with the stromal infiltration and desmoplasia of sclerosing BACs and CPAs.


Granulomatous endometritis associated with histologically occult cytomegalovirus in a healthy patient. This report describes the case of a 32-year-old female with chronic pelvic pain who was otherwise in good health. Endocervical curettings contained rare cells with intranuclear and cytoplasmic inclusions characteristic of cytomegalovirus (CMV) infection. Endometrial curettings demonstrated a stromal lymphocytic and plasmacytic infiltrate as well as numerous small, non-necrotizing granulomas, but no CMV by microscopic examination. However, CMV was identified by the polymerase chain reaction in DNA extracted from a paraffin section of the endometrial tissue. In conjunction with previous reports, the clinical and pathologic features of this case suggest that CMV can cause chronic endometritis in nonimmunocompromised patients. Furthermore, CMV infection should be considered in the differential diagnosis of granulomatous endometritis. This case demonstrates the usefulness of using the polymerase chain reaction to detect CMV in paraffin-embedded material.


Functional activity of protein C in newborn infants. A report of a study and a review of the literature. Protein C is lower in newborn infants than in adults. There are conflicting reports regarding functional activity and the presence of des-gamma carboxylated species in the newborn. We have compared protein C activity and antigenic level in newborn infants and found the activity: antigen ratio to be lower than in adults (0.69 versus 1.0). We discuss this finding in relation to the previously published reports.


Vasoactive intestinal peptide receptor expression on human lymphoblasts. This study was designed to determine which (if any) subtypes of leukemic blasts express a functional receptor for vasoactive intestinal peptide (VIP). Blasts harvested from bone marrow of 38 newly diagnosed patients were classified as acute lymphocytic leukemia (CALLA + pre-B-cell leukemia, CALLA-, pre-B-cell leukemia, T-cell leukemia) or acute myeloid leukemia based on cytochemical and histochemical markers. Of the 32 patients with lymphocytic leukemia, 22 expressed the VIP receptor as evidenced by VIP-mediated activation of adenylate cyclase in cell homogenates. Binding of 125I-VIP to ALL cells correlated with the ability of VIP to activate adenylate cyclase. The VIP receptor was not identified in myeloid blasts from any of six patients. Further correlation of 125I-VIP binding and VIP-mediated stimulation of adenylate cyclase was demonstrated in transformed cell lines: a pre-B-cell line (Nalm 6) and a T-cell line (Molt 4b) exhibited high-affinity binding of 125I-VIP and VIP-mediated activation of adenylate cyclase, whereas neither the histiocytic line (U937) nor the myelocytic line (HL60) appeared to express the VIP receptor. These observations suggest a role for VIP in the proliferation or differentiation of human T and B lymphocytes.


gamma-Hydroxybutyric acid in the treatment of alcohol dependence: a double-blind study. The effect of gamma-hydroxybutyric acid on alcohol consumption and alcohol craving in alcoholics was investigated in a randomized double-blind study versus placebo. Patients were treated as outpatients during a three month period either with gamma-hydroxybutyric acid (50 mg/kg/day, divided into three daily doses) or with placebo. Of the 82 alcoholics that entered the study, 71 completed it, 36 in the gamma-hydroxybutyric acid and 35 in the placebo group. Alcohol consumption was assessed by the subject's self report. At the 3rd month of treatment, 11 patients in the gamma-hydroxybutyric acid group referred to be abstinent and 15 referred controlled drinking; while in the placebo group only two and six patients referred abstinence and controlled drinking, respectively. Serum-gammaglutamyl-transferase activity correlated with the admitted alcohol consumption. Gamma-hydroxybutyric acid treatment decreased alcohol craving during the 3 months of treatment. Transient side effects were noted by six patients on gamma-hydroxybutyric acid and two on placebo. The results suggest that gamma-hydroxybutyric acid may be useful in the treatment of alcohol dependence.


Chronic alcohol intake induces reversible disturbances on cellular Na+ metabolism in humans: its relationship with changes in blood pressure. The effect of chronic alcohol consumption on Na(+)-K+ ATPase, Na(+)-Li+ countertransport, outward Na(+)-K(+)-Cl- cotransport system and the Na+ leak was investigated in red blood cells from 18 normotensive subjects with a daily alcohol intake of more than 150 g. The study was repeated after 3 months of alcohol withdrawal, and results were compared with a group of 20 healthy normotensive teetotalers. Maximal efflux rate (Vmax) and apparent dissociation constant for internal Na+ (KNa) of the Na(+)-K+ pump and the Na(+)-Li+ countertransport were significantly higher in alcohol consumers. A positive correlation between daily alcohol intake and Vmax of both transport systems (p less than 0.05) was observed. These values significantly decreased after alcohol withdrawal. A simultaneous stimulation of the Na(+)-K(+)-Cl- cotransport system after alcohol withdrawal was also observed. Blood pressure values were higher in alcoholics (133.7/82.3) than in abstainers (121.4/75 mmHg) and significantly decreased (128.5/76.9 mmHg) after withdrawal. A positive correlation between the stimulation of the Na(+)-K(+)-Cl- cotransport and the decrease of blood pressure after withdrawal was observed. In conclusion, chronic alcohol intake induces disturbances on red blood cell Na+ metabolism that dissipate with the cessation of drinking. Similar abnormalities also reported in humans and animals with primary hypertension have been associated in the pathogenesis of essential hypertension. Therefore, the pressor effect of chronic alcohol intake could be mediated through these changes in cellular Na+ metabolism.


Systolic blood pressure and neuropsychological test performance of alcoholics. This study examined resting systolic blood pressure (SBP) as a mediator of neuropsychological (NP) test performance in nonhypertensive alcoholics and controls. A median-split was used to assign alcoholics and controls to low and high SBP groups. Results showed that: (1) When SBP level was not considered, alcoholics only performed worse than controls on the WAIS Comprehension subtest. (2) Alcoholics and controls in the high SBP groups had fewer correct items on the WAIS Comprehension and Raven's, Set I tests than subjects in the low SBP groups. (3) Significant Diagnostic Group by SBP Group interaction was found for the Sentence Writing test. For this test only controls in the High SBP Group did worse than controls in the Low SBP Group. (4) Individual group comparisons for all NP tests showed that alcoholics in the High SBP Group were more impaired than controls in the Low SBP Group on the WAIS Comprehension, Shipley Abstraction Age and Stark Visual-Spatial tests; but alcoholics in the Low SBP Group did not differ from, or outperformed, controls in the High SBP Group on the WAIS Comprehension, Shipley Abstraction Age, Raven's, Set I, and Stark Visual-Spatial tests. These data demonstrate that both alcoholism and high SBP adversely and differentially affect the NP test performance of alcoholics and controls.


Modulation of f-met-leu-phe induced chemotactic activity and superoxide production by neutrophils during chronic ethanol intoxication. Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.


Effects of isoflurane on K+ and Ca2+ conductance in isolated smooth muscle cells of canine cerebral arteries. Although isoflurane is a known cerebral vasodilator, the mechanism of isoflurane-induced vasodilation is not clear. The purpose of this study was to investigate the effects of 2.6% isoflurane (1.2 mM) on macroscopic calcium and potassium channel currents in voltage-clamped canine middle cerebral artery cells. Cells were dialyzed with K(+)-glutamate solution and superfused with Tyrode's solution for measurement of potassium current (n = 20). Stepwise depolarization from a holding potential of -60 mV to beyond -30 mV elicited an outward, slowly inactivating potassium current that was reduced 50% +/- 2% and 81% +/- 3% (mean +/- SEM) in the presence of 1 mM 4-aminopyridine and 30 mM tetraethylammonium, respectively. Calcium ionophore (A23187, 10 microM) increased the potassium current by 76% +/- 3%, suggesting calcium dependency. Isoflurane reduced the amplitude of the potassium current by 35% +/- 4%. Calcium current was measured in cells dialyzed with solution containing 130 mM Cs(+)-glutamate and superfused with solution containing 10 mM BaCl2 and 135 mM tetraethylammonium to pharmacologically isolate the calcium current (n = 13). Under these conditions, progressive depolarizing steps from -60 mV elicited an inward current that was maximally activated at +20 mV and essentially eliminated by 1 microM nifedipine. This current, resembling a long-lasting (L-type) Ca2+ channel current, was reduced 40% +/- 4% by isoflurane. The results of this study suggest that isoflurane acts directly at the vascular muscle membrane to suppress transmembrane calcium and potassium currents. The decrease in calcium current would cause vasodilation; however, the concomitant decrease in potassium current may partially antagonize the depressant effect of isoflurane mediated through calcium current reduction.(ABSTRACT TRUNCATED AT 250 WORDS)


The classification of asbestos fibres by scanning electron microscopy and computer-digitizing tablet. Because of the need to completely and accurately size asbestos bulk samples for toxicity studies, a method was developed to classify asbestos fibres using enlarged micrographs originally produced on the scanning electron microscope (SEM). Individual fibre length and width measurements were performed on a computer-assisted, digitizing tablet. This method, though time consuming, permitted the sizing of all fibres (length and width) and particles (area) in selected fields of view at SEM magnifications of x100 and x2500. Final enlargement of the micrographs permitted sizing magnifications of x10,000. Seven distinct asbestos samples were classified including five chrysotile and two crocidolite samples. Statistical analyses showed good interfilter fibre size correlation for all types of asbestos. In addition, it was determined that a representative sizing of all fibres and non-fibrous particles on a filter preparation could be performed using five sets of micrographs taken at a magnification of x2500 and enlarged to x10,000: an inverse square root transformation (-0.5 power) is needed to normalize the distributions of length and width.


[The efficacy of transarterial immuno-embolization therapy in patients with hepatocellular carcinoma]. The efficacy of transarterial immuno-embolization therapy (TIE) was investigated in 7 patients with multiple hepatocellular carcinoma (HCC). We administered OK-432 (10 KE), fibrinogen (30 mg/ml) and thrombin (1 U/ml) as a new treatment for HCC with intrahepatic metastasis. In all patients with a high level of AFP, the AFP level decreased promptly less than the pretreatment level after TIE therapy. High fever in all cases, epigastralgia in 6 cases and appetite loss in 3 cases were observed after TIE therapy. Our results indicate that this therapy may be a safe and effective method in HCC patients with intrahepatic metastasis.


[Immunochemotherapy of carcinomatous peritonitis and pleuritis--report of 2 cases]. Intraperitoneal and pleural immunotherapy has been used as an effective therapy for malignancy. Recently we treated two patients with peritonitis and pleuritis due to cancer by intraperitoneal and pleural administration of IFN-gamma, OK-432 and antitumor agents. One patient with gastric cancer (stage IIIb) was treated with intraperitoneal administration of IFN-gamma and OK-432 in combination with intraarterial infusion of MMC, ADM, 5-FU and CDDP. Two months later, ascites and pleural fluid diminished. Another patient with ovarian carcinoma (stage IV), was administered IFN-gamma, OK-432 and CDDP into ascites with general medication of CDDP and Epi-ADM. Two months later, her ascites and tumor size decreased. This patient was treated with palaplatin every two months for the ten months and hysterosalpingecctomy and tumorectomy of Douglas pouch were performed at the sixteenth month. The histopathological examination of resection from this patient showed complete necrotic tissue of tumor. Endogenous cytokine therapy with intraperitoneal and pleural administration of IFN-gamma for priming and OK-432 for eliciting in combination with antitumor agents may be effective treatment for malignant peritonitis and pleuritis.


[Effects of intra-arterial infusion chemotherapy with radiation therapy for advanced hepatocellular carcinoma]. Eight cases were treated with periodic arterial infusion therapy from reservoir and four cases with arterial infusion therapy plus 30 Gy's radiation therapy for hepatocellular carcinoma (HCC). The anatomical extent of the tumor was E3 and E4, respectively. We evaluated these forms of therapy from the viewpoint of tumor characteristics, survival time and therapeutic effects. There was no effective case of life prolongation and most of the reservoir only treated cases died a short time after therapy. But no severe complication was observed in reservoir plus radiation cases. One case showed a response, and another case tumor necrosis. The results indicate that this method is effective for advanced HCC.


[Intra-arterial infusion chemotherapy for hepatocellular carcinoma using an implantable drug delivery system]. Intra-arterial chemotherapy and chemoembolization using implantable drug delivery system were performed in sixty-one cases of unresectable hepatocellular carcinoma (HCC). The following results were obtained. (1) The follow-up period was 253 days, and the administrations numbered 13.9 times on average. (2) Response rate was 31.1%. (3) AFP decreased in 80% cases, and PIVKA-II in 83.3% cases. (4) Tumor thrombus in the portal vein disappeared in 4 of 40 cases(10%). (5) Free-CDDP continued for 60 minutes after injection of cisplatin-phosphatidyl-choline-Lipiodol (CPL), and the response rate was 47%. (6) The survival rates were 56% in one year, 25% in two years, and 20% for three years. These results were significantly higher than those of one shot therapy. It is suggested that good therapeutic effects can be obtained by the implantable drug delivery system, and CPL was a useful anticancer agent for the therapy.


["Wrapping therapy" for advanced hepatic cancer with arterial and intraportal infusion chemotherapy]. A new treatment method to intercept collaterals using silicone rubber sheeting was used for 5 patients with advanced hepatic cancer. The therapy was carried out to prevent new collaterals. This procedure was followed by arterial chemoembolization, arterial infusion chemotherapy and intraportal infusion chemotherapy. The results were complete response in 2 patients, partial response in 2 patients, and no change in 1 patient. The overall survival time was 7-54 months after wrapping. Although a randomized control study is necessary to assess the true value of this method, the new therapy is considered worth using as an adjuvant treatment for advanced hepatic malignancies uncontrolled by arterial chemotherapy or chemoembolization.


[Prophylactic chemotherapy by regional arterial infusion in resected hepatoma patients]. We established risk factors for high post-resectional recurrence in patients with hepatocellular carcinoma (HCC). They included: (1)Vp (+), (2)IM (+), (3)more than 5 cm in diameter of the main tumor, and (4)gross type except single nodular tumor. Seventy HCC patients in this category were divided into two groups. In group 1, 11 patients prophylactically underwent hepatic arterial infusion chemotherapy after liver resection. Chemotherapeutic agents (MMC, 5-FU, ADM, CDDP) with Lipiodol were administered 4 times a year via Infuse-A-port. The remaining 59 cases served as the control without prophylactic infusion. Two-year survival rate was better in prophylactic group (75%) than in the control (46%, p = 0.063). The two-year disease-free survival was significantly improved in group 1 (40%) compared with that in group 2 (26%, p = 0.019). Based on our data, we suggest that prophylactic arterial infusion chemotherapy can be efficacious in alleviating hepatoma recurrence after liver resection.


[Postoperative arterial infusion chemotherapy for hepatocellular carcinoma]. We performed arterial infusion chemotherapy by injection of adriamycin plus Lipiodol after radical hepatectomy in patients with hepatocellular carcinoma for prevention of tumor recurrence. The clinical studies showed that the 1-, 2-, 3-, 4-, and 5-year cumulative disease free rates were 87.5, 73.4, 52.4, 37.7% and 37.7% in patients with this chemotherapy, and 76.1, 46.5, 30.4, 30.4% and 30.4%, respectively in those without this chemotherapy, indicating a significant difference (p less than 0.05) between these two groups. This method of chemotherapy was particularly effective for patients with vascular involvement, intrahepatic metastasis or tumors more than 3cm in diameter.


[Prevention of postoperative recurrence of hepatocellular carcinoma by adjuvant locoregional chemotherapy]. Postoperative adjuvant locoregional chemotherapy was performed on patients who underwent hepatic resection for hepatocellular carcinoma with permeation of tumor into portal vein and/or into hepatic vein and/or intrahepatic metastasis to prevent recurrence. Twenty patients received intra-hepato-arterial infusion of chemotherapeutic drugs or Lipiodol, or transcatheter arterial embolization as adjuvant therapy, and 25 patients did not receive the therapy. There was no significant difference in the recurrence rate between the two groups, and disease-free-survival would not be improved significantly by adjuvant therapy. But the disease-free-survival was improved in patients who underwent relative noncurative hepatectomies. Median duration from the hepatic resection to the recurrence was prolonged by adjuvant therapy, but no significant difference was noted between the two groups. Serious hepatic injury was a side effect of adjuvant therapy in one patient. Therefore great care was necessary when performing the therapy. Further study on choice of the drugs and methods was considered necessary to obtain more effective therapy.


[Intermittent intra-arterial chemotherapy using subcutaneously implanted reservoir for hepatocellular carcinoma and liver metastasis of colorectal carcinoma]. Hepatectomy has been performed as a treatment for hepatocellular carcinoma (HCC) and metastatic liver carcinoma. The prognosis has improved, but it is not sufficient. In our department hepatic arterial infusion chemotherapy using subcutaneous implanted reservoir has been undertaken in 21 of 66 patients who underwent hepatectomy in hepatocellular carcinoma and 13 of 30 in liver metastasis of colorectal carcinoma since 1986. There was no significant difference between the group with and without arterial infusion chemotherapy in HCC but there was a significant difference in metastasis of colorectal carcinoma. In unresectable cases, intra-arterial chemotherapy was undertaken, but there was no significant difference. On the other hand, 26 of 58 cases receiving arterial infusion chemotherapy have shown complications.


[Disappearance of lung metastases from hepatocellular carcinoma following bronchial arterial infusion of CDDP and MMC]. A 58-year-old man was admitted to our department because he had been diagnosed as hepatocellular carcinoma, which was located at S6 segment, and posterior segmentectomy was performed. After 6 months, right lung metastases of HCC were found and right bronchial arterial infusion of CDDP and MMC was performed twice. Dramatic effects were obtained such as disappearance of lung metastases. We emphasize the useful effect of CDDP and MMC for metastases of HCC.


[Two cases of esophageal varices complication after hepatic arterial infusion chemotherapy (HAI) for metastatic liver tumor]. Mild liver dysfunction is a well-known complication of HAI, but it has been thought to be transient and reversible in most cases. In the case, of metastatic liver disease, in particular, HAI has been performed safely because liver function is normal for the most part. We encountered 2 cases of irreversible severe liver dysfunction and esophageal varices after hepatectomy for metastatic liver tumor from colorectal cancer. They were treated with postoperative adjuvant HAI. Biliary enzyme as alkaline phosphatase elevated, and dilated intrahepatic bile ducts were observed in both patients. Fibrosis of Glissonean sheath, dilatation of intrahepatic bile ducts and intrahepatic biliary stones were observed at autopsy in both patients. One of the patients had obstruction of portal trunk. It must not be forgotten that such complications can occur even in a case with non-cirrhotic liver.


[Successful preparation of mitoxantrone emulsion containing non-ionic contrast medium]. Mitoxantrone, a new anti-cancer agent, was successfully prepared for Lipiodol emulsion. The mixture of Mitoxantrone and non-ionic contrast medium, Omnipaque 300, was combined with Lipiodol at the ratio of 1:2. When the ratio of Mitoxantrone and Lipiodol was 1:4, microscopic study revealed stabilized water in oil emulsion, which could release the anti-cancer agent slowly. We applied it for a case of hepatocellular carcinoma with good result. Intra-arterial infusion of this emulsion might be considered effective for treatment of hepatocellular carcinoma.


[Problem of the long-term arterial infusion therapy with implantable reservoir for unresectable hepatocellular carcinoma]. Arterial infusion therapy with implantable reservoir was performed for forty-seven cases with unresectable hepatocellular carcinoma since 1986-1991. The one-year survival rate of cases of arterial infusion therapy with implantable reservoir was 50.6% and the 2-year survival rate was 37.8%. The survival rate was 51.4% and 32.2%, respectively, in the TAE cases for the same period. Considering the kinds of reservoirs used for HCC, the survival curve of 23 cases with double lumen reservoir showed a significantly better pattern than that of 24 cases with the usual single lumen reservoir (p less than 0.05). Cumulative usable rate of reservoir was 78.6% at one year, and 42.4% at two years (Kaplan-Meier method). Therapy after the reservoir was unusable, particularly with arterial obstruction by catheter, was restricted because arterial infusion therapy was impossible. It is considered very important to retain the hepatic artery because arterial infusion therapy is mainly for unresectable hepatocellular carcinoma.


[A device for hepatic arterial catheterization using saphenous vein graft]. We performed regional arterial infusion chemotherapy for hepatocellular carcinoma by retaining a hepatic arterial reservoir. The catheters were placed via gastroduodenal artery and positioned at the junction with common hepatic artery during operation. But we encountered many complications and did not perform regional arterial infusion chemotherapy. This time we cannulated through the saphenous vein graft by which hepatic perfusion was maintained by the superior mesenteric artery; we did not cannulate through the common femoral artery using a subcutaneously implanted reservoir. Venous arterial anastomosis is performed with interrupted (7-0 Prolene) vascular suture. Postoperative angiography by the reservoir showed total hepatic perfusion. There have been no instances of hemorrhage, thrombosis, or catheter dislodgement with this technique. Duration of follow-up varied from 2 to 7 months.


[Experimental study on the effect of injection with anticancer agent-oil suspension]. Local injection of an anticancer agent guided by endoscopy is thought to be effective for cancerous lesion associated with lymph node metastasis, if the anticancer drugs are drained into the lymph nodes. In the experimental study, anticancer drug-oil (nimustine-Lipiodol) (N-L) suspension (5 mg/ml) was injected into the tumor (Lewis lung cancer) that had been implanted sub dorsally in mice (57 black/6 mice) for the purpose of finding out the antitumor effect on the primary lesion. Then it was injected into sarcoma-180 that had been implanted into hind feet of mice (ICR mice) for the purpose of finding out the antitumor effect on metastatic lymph nodes. The results showed that the N-L suspension was effective for the primary cancerous lesion and metastatic lesions.


[Fundamental study on hyperthermic chemotherapy using adriamycin-loaded hydroxyapatite]. We developed a porous hydroxyapatite ceramic (HAP) incorporating adriamycin (ADM), that is, ADM-HAP as a new delivery system (DDS) to release ADM gradually. We also researched the possibility of hyperthermic chemotherapy using ADM-HAP by in vitro and in vivo experiments. As for in vitro experiments, we implanted HAPs into uniform Agar-Phantom, and observed thermal distribution generated by Thermotron-RF 8 using thermography. Then we found the hot spot that the edge temperature of HAPs always at the range of 0.5-0.7 degrees C than in the other regions. On the other hand, slow constant release (1%) of ADM from ADM-HAP in PBS was recognized for 24 hrs up to 30 days. When the incubating temperature was shifted up to 42.5 degrees C or 44 degrees C from 37 degrees C, the quantity released over 24 hrs increased about 1.1-fold or 1.3-1.4-fold of the cases at 37 degrees C, respectively. In the in vivo experiment, we inoculated Sarcoma 180 cells in the leg of ddY-mice, and measured the tumor growing times by the treatment of hyperthermia+ADM (whole body), hyperthermia+ADM (tumor region) or hyperthermia+ADM-HAP (tumor region). Then we found that the effect of hyperthermia with ADM-HAP inhibited synergistically the tumor growth as compared with hyperthermia with ADM. Consequently, we succeeded in tumor growth inhibition by increasing the temperature and by limiting ADM release to only a target region using hyperthermia with ADM-HAP.


[Stage III, IV hepatocellular carcinoma patients surviving more than five years after transcatheter chemoembolization]. Twelve of the patients with advanced (Stage III or IV) hepatocellular carcinoma who received transcatheter chemoembolization survived for more than 5 years. These survivors consisted of 6 Stage III cases and 6 Stage IV cases. Of the 6 Stage III cases, 5 had multiple tumors (larger than 2 cm) in one lobe of the liver, and 1 had tumor invasion into the second portal branch before treatment. Of the 6 Stage IV cases, 4 had had multiple tumors in both lobes, and 2 had had tumor invasion into the main portal branches. Before treatment, 9 of the 12 survivors had been rated as Child's class A. Lipiodol was used in all 12 cases. The results of the present study suggest the usefulness of Lipiodol in treating advanced hepatocellular carcinoma.


[Therapeutic difference by TAE between HCC with HCV-Ab and HBV-Ag]. Transarterial infusion therapy using adriamycin-Lipiodol emulsion (TAE) was used for 30 patients of HCC with HCV-Ab and 20 patients with HBV-Ag. We compared the tumor effect and prognosis in terms of several clinico-pathological factors. The response rate (PR+MR) after TAE was 43% in HCC patients with HCV-Ab and 30% in those with HBV-Ag. One-year survival rate was 89% in HCC patients with HCV-Ab and 58% in HCC patients with HBV-Ag. Thus, there was a significant difference between the two groups. No definite reasons between two groups influencing tumor effect and prognosis is obviously revealed except for portal vein invasion.


[Regional therapy to prevent recurrence after surgery in hepatocellular carcinoma]. The significance of regional therapy to prevent recurrence after surgery for hepatocellular carcinoma (HCC) was evaluated. In 275 patients who underwent hepatic resection for HCC, 143 (52%) had recurrences. Post-recurrence survival of the patients with regional therapy for recurrent foci was significantly better than that of patients without such therapy. Five-year post-recurrence survival of the patients treated with second surgery (77%) was significantly better than that of patients treated with chemoembolization (17%). The difference in 5-year survival between the former and the latter seemed to be caused by the difference in the degree of tumor progression at the recurrence. A second hepatic resection was the treatment of choice for a solitary intrahepatic recurrent tumor. However, chemoembolization was recommended for two or more recurrent foci.


[Effect of deprotonation on the conformational state of the tetracycline molecule]. Conformation rearrangements in the tetracycline molecule under the effect of the aqueous medium pH were investigated by circular dichroism and absorption spectrophotometry. Possible causes of the changes in the spectrum after the molecule deprotonation are discussed. An increase in the pH value was accompanied by folding-unfolding of the A-ring of the tetracycline molecule and its transfer within the pH neutral ranges to the conformation close to planar one and after increasing of the aqueous solution polarity to the folded conformation. The BCD chromophore did not change its initial flat conformation during deprotonation. The loss of the proton (4) by the nitrogen atom was accompanied by changes in the A chromophore chirality.


[Effect of penicillin precursors on antibiotic biosynthesis in various strains]. The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.


[Isosorbide dinitrate inhibits in vitro platelet aggregation at submicromolar concentrations]. Nitrate derivatives have in vivo and in vitro platelet anti-aggregant properties in addition to their vasodilatory effects. The mode of action is related to increased intracytoplasmic cyclic GMP concentrations. It has been shown that isosorbide dinitrate (ISDN) has this type of platelet anti-aggregant activity but the reported results about the active concentrations and the inhibited pathways of activation are contradictory. This study was designed to determine whether ISDN has in vitro platelet anti-aggregant activity at low doses and to verify if this effect is selective by aggregation induced by ADP. Finally, a possible potentialisation of the inhibitors due to ISDN was looked for with cyclic nucleotide phosphodiesterase inhibitors and with agents simulating the effect of adenylate cyclase. The results showed that: 1) ISDN had platelet anti-aggregant activity in vitro at concentrations of about 10-7 M, 2) that this effect was not limited to the aggregation induced by ADP as the aggregation induced by PAF-acether was also inhibited by low dose ISDN, 3) of the cyclic nucleotide modulators tested, only quercetine (flavonoide) potentialised the effects of ISDN.


[Mechanism of cellular action of nitrate derivatives]. Nitrate derivatives have to undergo metabolic activation in the smooth muscle cell or in the plasma with a sulflydryl radical. This transformation results in the formation of nitric oxide and/or S-nitrosothiols. These products stimulate an enzyme, the soluble guanylate cyclase in the sarcoplasm of the smooth muscle cell; giving rise to the formation of intracellular cyclic GMP from GTP. The cyclic GMP activates a kinase protein which in turn activates a number of other protein enzymes involved in the recaptation of calcium by the sarcoplasmic reticulum and in the extrusion of calcium from the cell. In addition, cyclic GMP reduces the level of phosphorylation of the myosin light chain, thereby reducing the sensitivity of the contractile proteins to intracellular calcium. All these phenomena cause smooth muscle relaxation so explaining most of the vasodilator effect of nitrate derivatives.


[Intracardiac metastases of primary neuroectodermal tumor]. Cardiac metastases from malignant tumors are rare in children and are usually found only at necropsy. A girl aged 6 months developed a swelling in the area of her left shoulder. Surgical excision was incomplete and pathological examination indicated neuroectodermic soft-tissue tumor. Chemotherapy with cyclophosphamide, doxorubicin then vincristine and actinomycin D was instituted, followed by second course of cyclophosphamide-doxorubicin. Three months after cessation of chemotherapy, the child developed manifestations of congestive cardiac failure that could not be induced by anthracycline (total cumulative dose: 275 mg/m2). Cardiac ultrasonography showed a metastatic tumor (diam = 3 cm) on the wall of the right ventricle and atrium, which was pedunculated inside the infundibular chamber. It was partly excised, and the patient was treated with etoposide and carboplatin. But a second metastatic tumor formed in the paravertebral area. This is the first description, to our knowledge, of cardiac metastases from neuroectodermic tumor. They were diagnosed by cardiac ultrasonography, as this imaging technique differentiates this complication from the anthracycline-induced cardiomyopathy usually seen in children developing cardiac failure under chemotherapy.


Role of atrial natriuretic peptide on calcium channel mechanisms involved in catecholamine release from bovine adrenal medulla. The role of the atrial natriuretic peptide on calcium channel mechanisms involved in catecholamine release was studied in the perfused bovine adrenal medulla. The atrial natriuretic peptide (1 nM and 10 nM) did not modify the spontaneous release of catecholamines. Ten nM of atrial natriuretic peptide decreased the output of catecholamines induced by acetylcholine, KCl-depolarizing solutions and angiotensin II. It was ineffective to modify the catecholamine release when calcium channels were blocked or in the presence of calcium-free media. Moreover, the deprivation of the ion calcium in the media decreased the catecholamine release induced by KCl to a lowest level, despite the presence of atrial natriuretic peptide in the perfusate. In conclusion, atrial natriuretic peptide inhibited the induced secretion of catecholamines in the bovine adrenal medulla and interfered as a partial blocker with calcium-dependent mechanisms.


Nor-binaltorphimine: a potent and selective kappa-opioid receptor antagonist with long-lasting activity in vivo. The kappa-receptor selectivity of nor-binaltorphimine (nor-BNI), a highly selective kappa-opioid receptor antagonist in vitro, was examined in vivo by measuring the time course of the antagonistic action of nor-BNI (5 and 20 mg/kg, s.c.) against the responses to U-50488H (10 mg/kg, s.c.), morphine (10 mg/kg, s.c.) and fentanyl (50 micrograms/kg, s.c.) in mice. In the tail pinch test, nor-BNI partially antagonized morphine and fentanyl analgesia, but not U-50488H analgesia in the first 30 min after s.c. administration. However, the kappa-antagonistic action gradually increased, reaching a plateau at 2 hr. This antagonistic action was maintained for at least 4 days. In contrast, the mu-antagonistic action declined to the control level at 2 or 4 hr after nor-BNI administration. In the acetic acid-induced writhing test, nor-BNI also exerted a more potent and selective kappa-antagonistic action at 24 hr than at 1 hr after its s.c. administration. Nor-BNI also showed a long-lasting kappa-antagonism against the hyperthermic response induced by U-50488H (5 mg/kg, s.c.). Thus, we found that nor-BNI is a slow-onset, long-lasting, selective kappa-antagonist in vivo.


Acetylene, a mammalian metabolite of 1,1,1-trichloroethane. 1,1,1-Trichloroethane (TCE) is a widely used industrial solvent of low acute toxicity. It is slowly oxidized to trichloroethanol and trichloroacetic acid by cytochrome P-450-dependent mono-oxygenases. Increased inhalative uptake by rats under hypoxia and spin-trapping experiments indicate that TCE is also reductively metabolized to a radical intermediate. Acetylene is formed as a metabolite, suggesting transfer of an additional electron to form the corresponding carbene. Hypoxia and induction of mixed-function mono-oxygenases accelerate the formation of acetylene. Experiments performed in vitro with rat liver microsomal fractions yield analogous results.


E.p.r. and magnetic circular dichroism spectroscopic characterization of bacterioferritin from Pseudomonas aeruginosa and Azotobacter vinelandii. The e.p.r. and magnetic circular dichroism (m.c.d.) spectra of bacterioferritin (BFR) extracted from Pseudomonas aeruginosa and Azotobacter vinelandii have been studied over a wide temperature range down to liquid-helium temperature. The e.p.r. spectra show the presence of low-spin Fe3+ haem with g values of 2.86, 2.32, 1.48 (P. aeruginosa) and 2.88, 2.31, 1.46 (A. vinelandii), in both the presence and absence of the BFR core. Together with evidence from the porphyrin-to-Fe3+ charge-transfer band at 2240 and 2270 nm the axial haem ligands are identified as two methionines. The low-temperature m.c.d. spectra in the region 300-1000 nm of P. aeruginosa and A. vinelandii BFR are identical with one another and unaffected by removal of the iron core. Hence it can be concluded that the presence of the iron core has no detectable effect on the electronic states and on the stereochemistry of the haem group. This was unexpected, in view of the observations by Watt, Frankel, Papaefthymiou, Spartalian & Stiefel [(1986) Biochemistry 25, 4330-4336] that the redox potential of the haem group in A. vinelandii BFR shifts from -475 mV to -225 mV on removal of the core. The e.p.r. spectra of holoBFR show a broad symmetrical derivative-shaped band centred at g = 2.0 which decreases in bandwidth as the temperature is raised. This signal is assigned to the uncompensated electron spins of the iron core.


Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.


Regulation of cyclic AMP synthesis and degradation is modified in rat liver at late gestation. Cyclic AMP (cAMP) is known to play a key role in regulating insulin action, and it is well documented that in several cases of physiological insulin resistance its concentration is increased. Since late pregnancy in the rat is associated with liver insulin resistance, we have studied possible alterations of some cellular mechanisms regulating the cAMP metabolism. (1) Liver cAMP concentration was shown to be increased by some 30% and 50% at 18 and 22 days of pregnancy respectively, compared with virgins. (2) Basal adenylate cyclase activity was higher only in the 18-days-pregnant rat, and the forskolin-stimulated maximal activity was similar in the three groups of animals. (3) alpha s protein is decreased in term-pregnant rats; however, coupling between Gs and adenylate cyclase is only impaired in the 18-days-pregnant animals, and stimulation by glucagon is impaired in both groups of pregnant animals. (4) Gi-2 protein was shown to be unable to elicit the tonic inhibition of adenylate cyclase in pregnant rats, although it was only decreased at 22 days of gestation. The increased alpha i-2 level detected by immunoblotting at 18 days of gestation did not correlate with its decreased ADP-ribosylation, suggesting that the protein is somehow modified at this stage. (5) Pregnancy is associated with a decrease in membrane phosphodiesterase activity. Our results show that late pregnancy is associated with increases in liver cAMP levels that might be involved in eliciting the characteristic insulin-resistant state, and suggest that mechanisms leading to these increments are changing during this phase of gestation.


Characterization of a Zn(2+)-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity. p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.


Angiotensin-converting enzyme of the human small intestine. Subunit and quaternary structure, biosynthesis and membrane association. Angiotensin-converting enzyme (ACE) was isolated from detergent-derived extracts of human intestinal brush-border membranes (BBMs) by immunoprecipitation using a monoclonal antibody. Analysis of the immunoprecipitates by SDS/PAGE revealed a polypeptide of apparent M(r) 184,000 under reducing and non-reducing conditions, indicating that ACE does not contain intermolecular disulphide bridges. The quaternary structure of ACE was examined using cross-linking experiments with dithiobis[succinimidylpropionate] (DSP) and density gradient centrifugation on sucrose gradients. Both approaches demonstrated that ACE is assembled in the membrane as a monomer. By contrast, the control glycoprotein aminopeptidase N (ApN) exists as a dimer. Biosynthetic labelling experiments in intestinal tissue explants demonstrated that the 184,000-M(r) protein is generated from a single-polypeptide, mannose-rich precursor of ACE (M(r) 175,000) by modification of the carbohydrate side-chains in the Golgi apparatus. The mode of association of the mature form of the enzyme with BBMs was investigated by hydrophobic labelling of right-side-out brush-border vesicles with the photoactivatable carbene-generating reagent 125I-labelled 3-(trifluoromethyl)-3-(m[formylamino]phenyl)diazirine (125I-labelled TID), followed by treatment with trypsin at dilutions that do not cause substantial degradation of ACE. These studies demonstrated that ACE is associated with the membrane via a hydrophobic segment. Furthermore, treatment of 35S-labelled inside-out membrane vesicles with trypsin revealed that ACE possesses a cytoplasmic tail, and therefore has a transmembraneous orientation.


Accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in thrombin-stimulated platelets. Different sensitivities to Ca2+ or functional integrin. Differences in regulation of the accumulation of PtdIns(3,4)P2 versus that of PtdIns(3,4,5)P3 were noted in thrombin-stimulated human platelets. The rapid (within 20 s) response of PtdIns(3,4,5)P3 contrasted with a distinct lag in the accumulation of PtdIns(3,4)P2 that was followed by a pronounced increase by 90 s. The presence of 2.5 mM-CaCl2 further elevated PtdIns(3,4)P2 by 50-120%, but only at a late stage (after 90 s). Tetrapeptide RGDS (Arg-Gly-Asp-Ser), which blocks the interaction of ligands such as fibrinogen with platelet integrin alpha IIb beta 3 (GPIIb-IIIa), inhibited only the late-phase PtdIns(3,4)P2 accumulation that was associated with added Ca2+. Although stimulated tyrosine phosphorylation of platelet protein (total cell lysate) was altered by Ca2+ or RGDS, we could not identify any such proteins that were affected comparably to PtdIns(3,4)P2. In contrast to the PtdIns(3,4)P2 response, the accumulation of PtdIns(3,4,5)P3 was unaffected by Ca2+ or RGDS at any time.


Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban. COS 1 cells were transfected with full-length pig stomach sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA)2a or SERCA2b cDNA. Ca2+ uptake by microsomes from transfected cells revealed that the Ca2+ affinity of the SERCA2b Ca2+ pump (K0.5 0.17 +/- 0.01 microM) was higher than that of the SERCA2a Ca2+ pump (K0.5 0.31 +/- 0.02 microM). Thapsigargin-sensitivity was found to be identical for the two isoforms. The Ca2+ affinity of both the SERCA2a and SERCA2b Ca2+ pumps was decreased by a factor of two when they were co-expressed with phospholamban.


Activation of neuromedin B-preferring bombesin receptors on rat glioblastoma C-6 cells increases cellular Ca2+ and phosphoinositides. Recent cloning studies confirm the presence of two subtypes of bombesin (Bn) receptors. In contrast to the gastrin-releasing peptide (GRP)-preferring subtype, which has been widely studied, nothing is known about the cellular mechanisms of the neuromedin B (NMB)-preferring subtype, which occurs widely in the central nervous system and gastrointestinal tissues, partially because of the lack of a cell line with functional receptors. In the present study we have investigated Bn receptors on the rat glioblastoma cell line C-6, reported to contain mRNA of the NMB receptor subtype. Binding of 125I-[D-Tyr0]NMB to these cells was time- and temperature-dependent, saturable, reversible, and only inhibited by Bn receptor agonists or antagonists. For Bn receptor agonists the relative potencies were: NMB (1.7 nM) approximately equal to litorin (3 nM) greater than ranatensin (8 nM) greater than Bn (19 nM) greater than neuromedin C (NMC) (210 nM) greater than GRP (500 nM). These relative affinities were almost identical to those for the NMB receptor subtype on rat oesophageal tissue and for Balb 3T3 cells stably transfected with the NMB receptor subtype. These potencies differed from those for the GRP receptor subtype on rat pancreatic acini [Bn approximately equal to litorin (4 nM) greater than ranatensin, NMC, GRP (15-20 nM) much greater than NMB (351 nM)]. The relative potencies of four different classes of Bn receptor antagonists were compared. Results from C-6 tumour cells agreed closely with those for binding to the NMB receptor subtype on rat oesophageal tissue and in Balb 3T3 cells stably transfected with this receptor, and differed markedly from those for binding to the GRP receptor subtype on rat pancreatic acini. Four Bn receptor antagonists had a higher affinity for the GRP subtype ([D-Phe6]Bn-(6-13)ethyl ester (500 x), [D-Phe6][psi 13-14,Cpa14]Bn- (6-14) (70 x) (where psi 13-14 refers to the replacement of the -CONH- peptide bond between Leu13 and Met14 by -CH2NH2) [psi 13-14,Leu14]Bn, [D-Phe6]Bn-(6-13) propylamide (30 x)] and two had a higher affinity for the NMB subtype on C-6 cells and transfected cells ([D-Pro4,D-Trp7,9,10] substance P-(4-11) (9 x) and [Tyr4,D-Phe12]Bn (18 x)]. In C-6 tumour cells, Bn receptor agonists caused an increase in cytosolic Ca2+ and the generation of inositol phosphates. For both responses, NMB was more than 50-fold more potent than GRP. Neither NMB nor GRP increased cyclic AMP. These results demonstrate that the rat glioblastoma cell line C-6 possesses functional NMB-preferring Bn receptors, and agonist occupation activates phospholipase C, thus increasing cytosolic Ca2+ and inositol phosphate formation. Because the interaction of Bn-related peptides with C-6 cell receptors is identical with that reported in other tissues containing the mRNA for the NMB subtype, this cell line should prove useful in exploring further the cellular basis of action of the peptides that interact with this receptor in the central nervous system and various other tissues.


Effects of EGF on the mass of inositol 1,4,5-trisphosphate and SN(1,2)-diacylglycerol in freshly isolated rat hepatocytes: comparison with vasopressin. We measured the masses of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) in hepatocytes in response to both epidermal growth factor (EGF) and vasopressin. EGF at 25 nM did not alter Ins(1,4,5)P3 content of hepatocytes. However, the combination of 100 nM EGF concentration and incubation with lithium did increase Ins(1,4,5)P3 content. This increase was only one tenth of that elicited by vasopressin in parallel incubations. This finding resolves a controversy concerning the ability of EGF to increase Ins(1,4,5)P3 in hepatocytes, and argues against a role for phosphoinositide hydrolysis in EGF action in hepatocytes. Both EGF and vasopressin caused a rapid (30 s) increase in DAG content. A delayed increase in DAG content, that was maximal after several minutes, was observed only for vasopressin. The rapid increase in DAG content implies an activation of protein kinase C for both EGF and vasopressin.


Evidence for a new inositol phospholipid in rat brain mitochondria. Phosphorylation of phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and diacylglycerol (DAG) was studied in rat brain cortex myelin, synaptosomal and mitochondrial fractions, with ATP as phosphate donor and endogenous phospholipids as substrate. All fractions had PI, PIP and DAG phosphorylating activity with their own characteristic subcellular distribution. However, in the mitochondrial fraction an unidentified lipid was phosphorylated, which had a slower Rf value than PIP2 on TLC. After hydrolysis of the polar head group of the lipid and separation on anion exchange columns, it appeared to be a phosphoinositide. The elution profile showed that it was not phosphatidylinositol trisphosphate, or a lyso-compound. The available evidence suggests that the unknown inositol phospholipid in rat brain mitochondria is a phosphatidylinositol 4,5-bisphosphate isomer, although the possibility of it being a glycosyl-phosphoinositide cannot be excluded.


Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells. 1,25-dihydroxycholecalciferol stimulates membrane phosphoinositide turnover in colonic epithelial and other cells, but the effects of this hormone on phosphoinositide metabolism in specific antipodal plasma membranes has not been examined. In the present studies, addition of 10(-8)M 1,25-dihydroxycholecalciferol to rat colonic crypts for 90 seconds decreased the phosphatidylinositol-4,5-bisphosphate content and increased the diacylglycerol content of the baso-lateral, but not the brush border plasma membrane. Using Caco-2 cells grown as tight polarized monolayers, 1,25-dihydroxycholecalciferol reduced cellular phosphatidylinositol-4,5-bisphosphate and increased cellular inositol-1,4,5-triphosphate and diacylglycerol when added to the buffer bathing the baso-lateral, but not the brush border membrane surface. These data indicate, therefore, that 1,25-dihydroxycholecalciferol activates the phosphoinositol signal transduction cascade specifically in the baso-lateral cell membrane of colonic cells.


Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae). Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.


The endothelin-A receptor subtype transduces the effects of the endothelins in the anterior pituitary gland. All members of the mammalian endothelin family of peptides exert significant effects on prolactin and luteinizing hormone release from dispersed anterior pituitary cells in vitro. The rank order of potency for the prolactin inhibiting effects of the endothelins is ET-1 = ET-2 much less than ET-3. This suggests an involvement of the ET-A receptor subtype. The selective ET-A receptor antagonist BQ-123 antagonized the effects of the ETs in a competitive fashion with pA2 values of 6.1 (ET-1), 5.7 (ET-2) and 6.4 (ET-3), when added simultaneously with the ETs. This suggests the involvement of the ET-A receptor subtype in the actions of the ETs within the anterior pituitary gland.


Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis. delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.


Leukotriene B4 induced steady state calcium rise and superoxide anion generation in guinea pig eosinophils are not related events. We have examined the nature of the leukotriene B4 (LTB4) induced steady state intracellular calcium rise [Ca++]i in guinea pig eosinophils and the relationship between LTB4 induced [Ca++]i and superoxide anion (O2-). LTB4 induced a rise in intracellular Ca++ (following a Ca(++)- transient) in a dose dependent manner with an optimal increase around 50-100 nM. Depolarizing concentrations of K+ did not induce [Ca++]i in eosinophils nor did the voltage operated calcium channel inhibitor, nifedipine, inhibit the LTB4 induced Ca++ entry. In contrast, SK&F 96365 a purported receptor operated calcium channel (ROCC) inhibitor, and its parent compound SC 32849, attenuated LTB4 induced [Ca++]i. Five reference anti-asthmatics (ketotifen, formoterol, disodium-cromoglycate, theophylline and budesonide) had no influence on LTB4 induced [Ca++]i. LTB4 also induced O2- generation (a functional response) in a dose dependent manner with optimal effect around 100 nM. However, in contrast to Ca(++)- influx, LTB4 induced O2- generation was not affected by either SK&F 96365 or its analogues or reference anti-asthmatics. The results of this study suggest a) the presence of a non-voltage gated, receptor operated, calcium sequestration process in guinea pig eosinophils, b) that LTB4 induced [Ca++]i and O2- generation are apparently unrelated events in these cells, and c) that standard anti-asthmatics do not have an influence on either LTB4 induced [Ca++]i or O2- generation in these cells.


Synthesis of a biospecific adsorbent for the purification of the three human retinoic acid receptors by affinity chromatography. The total synthesis of an affinity gel suitable for the purification of retinoic acid receptors (hRARs) is reported. A chalcone derived from a potent retinobenzoic acid (Ch55) was chosen as the ligand and fixed to an immobilized matrix by coupling with the N-hydroxysuccinimide ester of agarose (Affi-Gel 10, Bio-Rad Laboratories). Efficiencies of purification of the different human RARs were tested, using recombinant receptors produced with the baculovirus expression system.


Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast. We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1). Such constructs could constitute convenient shuttle vectors between yeast and mouse cells. However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae. A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus. One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus. Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment. This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element. Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent. These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.


Expression of platelet activating factor receptor in renal tubular cell line (LLC-PK1). Northern blot analyses of poly(A)+ RNA extracted from cloned renal tubule cells, LLC-PK1, MDCK and JTC-12 cells, were performed using an isolated cDNA of guinea pig platelet activating factor (PAF) receptor as a probe. The results showed the expression of single entity of PAF receptor mRNA in LLC-PK1, but not in MDCK or JTC-12 cells. PAF also induced a dose-dependent elevation of intracellular calcium in LLC-PK1 cells as assessed by Fura-2 fluorescence signals, which was completely blocked by a PAF receptor antagonist, WEB 2170. These results indicate a PAF receptor-effector system in a renal tubular cell line LLC-PK1, providing a possible model system for studying physiological roles of PAF receptors in renal tubules.


The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody. The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.


Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors. We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.


Characterization by cDNA cloning of the mRNA of a highly basic human protein homologous to the yeast ribosomal protein YL41. From a cDNA library in lambda gt11 derived from poly(A)+ mRNA of human ovarian granulosa cells, a cDNA clone lambda HG12.1, containing an EcoRI insert of 470 bp, was identified. After subcloning of the insert into pUC18, the clone pHG12.1 was obtained and sequenced. The 5'-region of the insert of pHG12.1 was extended by the polymerase chain reaction (PCR) with cloned total cDNA. Assembly of the PCR fragment with the insert of pHG12.1 yielded clone pHG12. From the first open reading frame of pHG12 the amino acid sequence for a polypeptide of 25 amino acid residues (designated HG12) was derived, which was identical in 22 residues with yeast ribosomal protein YL41. It is therefore assumed that HG12 is the first mammalian homolog of yeast ribosomal protein YL41. Transcription of DNA fragments containing the coding region of pHG12 cloned into BluescriptM13, followed by cell-free translation, yielded a polypeptide with an apparent mol.wt. of 14.5 kDa, much larger than the theoretical mol.wt. (3454 Da). The discrepancy between theoretical and apparent mol.wt. was also observed for yeast ribosomal protein YL41. Southern analysis revealed that HG12 is not specified by a single copy gene. Homology for HG12 specific sequences is observed for bovine, porcine and rat species.


Genomic structure and expression of the human fau gene: encoding the ribosomal protein S30 fused to a ubiquitin-like protein. The fau gene is the cellular homolog of the fox sequence in the Finkel-Biskis-Reilly Murine Sarcoma Virus (FBR-MuSV). This virus acquired the fau sequence in its reversed transcriptional orientation. Human and mouse fau cDNA's were identified and both encode a new protein of 133 AA. We show that fau (for FBR-MuSV associated ubiquitiously expressed gene) becomes expressed in all different tissues tested as a 600 bp messenger and we report the genomic structure of the human fau gene. The gene consists of five exons and four introns and the 5' untranslated region displays characteristic features for a housekeeping gene. Fau encodes the ribosomal protein S30 fused to a Ubiquitin-like protein.


A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase. Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.


Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells. The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.


Inhibition of pig aortic smooth muscle cell DNA synthesis by selective type III and type IV cyclic AMP phosphodiesterase inhibitors. Foetal calf serum (FCS) and platelet-derived growth factor (PDGF)-stimulated incorporation of [3H]thymidine into pig aortic smooth muscle cell (ASMC) DNA was decreased by agents that either stimulated the synthesis (forskolin) or inhibited the breakdown (3-isobutyl-1-methylxanthine, IBMX) of cAMP. FCS-stimulated incorporation of [3H]thymidine into DNA was also reduced by selective inhibitors of cAMP-specific phosphodiesterase (PDE IV) (Ro-20-1724, rolipram) and cGMP-inhibited cAMP PDE (PDE III) (SK&F 94836). IBMX, Ro-20-1724, rolipram and SK&F 94836 enhanced forskolin inhibition of DNA synthesis. Alone, rolipram was a relatively weak inhibitor of FCS-induced ASMC DNA synthesis (IC25 greater than 20 microM); however, in the presence of a threshold concentration of SK&F 94836 (20 microM), the potency of rolipram increased (IC25 = 4 microM), suggesting synergy in the actions of PDE III and PDE IV inhibitors. SK&F 94836 and rolipram elicited 30% and 37%, respectively, reductions in FCS-induced ASMC proliferation and potentiated the inhibitory actions of forskolin. PDE III and PDE IV inhibitors alone, exerted minimal effects on ASMC cAMP levels after a short term (10 min) or long-term (2 or 24 hr) exposure, but enhanced forskolin-induced accumulation of cAMP. ASMC spontaneously released cAMP into the extracellular medium, a process that was increased by forskolin. PDE III and PDE IV inhibitors had no effect alone on cAMP extrusion but enhanced the effect of forskolin. Exposure of ASMC to forskolin or SK&F 94836 for 15 min increased the activity ratio (AR) of cAMP-dependent protein kinase from 0.05 to 0.17 and 0.23, respectively. Ro-20-1724, alone, did not affect cAMP-dependent protein kinase but enhanced the stimulatory effect of forskolin (AR = 0.37) and SK&F 94836 (AR = 0.27). Agents that increased cGMP synthesis (glycerol trinitrate, atrial natriuretic factor) or decreased its hydrolysis by selectively inhibiting cGMP-specific PDE (PDE V) (zaprinast) exerted no effects on FCS- or PDGF-stimulated [3H]thymidine incorporation into DNA either alone or in combination. The cytosolic fraction of pig ASMC contained four cyclic nucleotide PDEs which were categorized as PDE V, Ca2+/calmodulin-stimulated PDE (PDE I), PDE III and PDE IV. PDE I and III activities were also associated with the particulate fraction. The results demonstrate that inhibitors of PDEs III and IV alone or in combination with forskolin, reduce ASMC DNA synthesis and proliferation, through an action likely to involve elevation of intracellular cAMP. In contrast, inhibition of cGMP hydrolysing PDE subtypes (I and V) exerted no effect on DNA synthesis in this cell type.


Differential turnover of enzymes involved in human monocyte eicosanoid metabolism. Selective inhibition of cyclooxygenase product formation by cycloheximide in the absence of effects on 5-lipoxygenase or phospholipase A2. Human monocytes treated with cycloheximide (CHX) demonstrated a concentration- and time-dependent inhibition of prostaglandin E2 (PGE2) synthesis and release in response to stimulation with phorbol myristate acetate, ionomycin, serum-treated zymosan, or concanavalin A. The effect of CHX required preincubation and was largely reversible within 2 hr. Thromboxane A2 release was affected similarly but no comparable effects were observed on labeled arachidonic acid release or leukotriene B4 generation. The PGE2 response was also inhibited by CHX when monocytes were given exogenous arachidonic acid with or without stimulation. CHX pretreatment also comparably decreased the amount of immunoreactive cyclooxygenase in resting and stimulated monocytes. These data indicate that monocyte cyclooxygenase, in contrast to phospholipase A2 or 5-lipoxygenase and their regulatory proteins, turns over rapidly and may be a target for up- or down-regulation by pharmacologic or (potentially) physiologic agents which affect protein synthesis or degradation.


Catabolic disposition of 3'-azido-2',3'-dideoxyuridine in hepatocytes with evidence of azido reduction being a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides. 3'-Azido-2',3'-dideoxyuridine (AzddU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in vitro with low bone marrow toxicity. Although AzddU is currently being evaluated in clinical trials, its catabolic disposition is unknown. Pharmacokinetic studies in rhesus monkeys have demonstrated that a 5'-O-glucuronide is excreted in urine. The present study examined the catabolic disposition of AzddU is isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AzddU, low intracellular levels of two catabolites, identified as 3'-azido-2',3'-dideoxy-5'-beta-D-glucopyranosyluridine (GAzddU) and 3'-amino-2',3'-dideoxyuridine (AMddU), were detected. Studies using rat microsomes demonstrated that GAzddU formation was only detected in the presence of uridine 5'-diphosphoglucuronic acid, and that the rate of AMddU formation increased significantly in the presence of NADPH. Under similar conditions, reduction of the 3'-azido function was also demonstrated herein with 3'-azido-2',3'-dideoxycytidine (AzddC), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeC) and 3'-azido-2',3'-dideoxyguanine (AzddG), suggesting that enzymatic reduction to a 3'-amino derivative is a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides at the hepatic site.


Interaction of beta-eudesmol with Na+,K(+)-ATPase: inhibition of K(+)-pNPPase activity. The effect of beta-eudesmol, one of the major components in So-jutsu (Atractylodis Lanceae Rhizoma), on K(+)-dependent p-nitrophenyl phosphatase (K(+)-pNPPase) activity was studied. It inhibited K(+)-pNPPase activity with an I50 value of 4.1 x 10(-4) M. The inhibition rate decreased as the K+ concentration was increased, whereas greater inhibition was observed with high concentrations of either Na+ or ATP. The Ki values for Na+ in the presence of 0, 0.1 and 1 mM ATP were 140, 260 and 310 mM, respectively, but with the addition of beta-eudesmol, these values decreased to 90 mM regardless of the ATP concentration. This study on K(+)-pNPPase activity supports the conclusion obtained from the study on Na+,K(+)-ATPase activity (Satoh K et al., Biochem Pharmacol 44: 373-378, 1992) that is, beta-eudesmol interacts with enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P. Furthermore, in the study of the effects of K+ and beta-eudesmol on K(+)-pNPPase activity, it was confirmed that beta-eudesmol prevents the conformational change of Na.E1----K.E2.


Effect of moracizine and ethacizine on receptors of potential-operated calcium channels and calcium-binding proteins. The action of two antiarrhytmic drugs, moracizine (MOR, CAS 31883-05-3) and ethacizine (ETHA, CAS 33414-33-4) on receptors of potential-operated CA-channels has been investigated. ETHA binding to verapamil receptors was more effective than that of MOR (IC50 = 0.53 +/- 0.08 mumol/l, respectively). The Hill coefficient for ETHA binding was similar to that of verapamil (0.64 +/- 0.09 and 0.60 +/- 0.10, respectively). Interaction of ETHA and MOR with dihydropyridine receptors in concentrations up to 10 mumol/l was similar that of verapamil, however, MOR was less potent. MOR and ETHA did not interact with calmodulin and troponin C at concentrations up to 100 mumol/l. The influence of MOR and ETHA on enzymes dependent on Ca-binding proteins (phosphodiesterase and actomyosin ATPase) was not observed up to 100 mumol/l. Comparison of clinical and electrophysiological data with these results allows the conclusion that ETHA exerts Ca-blocking effects by the interaction with verapamil receptors on potential-operated Ca-channels.


[Histopathologic features of opportunistic infections of the small intestine in acquired immunodeficiency syndrome]. The frequency of the opportunistic infections of the duodenum in AIDS patients was determined by way of histologic study in 207 patients between January 1987 and June 1991. All cases had serial paraffin sections, run through HES, PAS, Giemsa, Brown-Brenn, and Zieh-Neelsen stains, and 20 cases had in addition cytologic and electron microscopic study. 63 patients had opportunistic infections (10 cryptosporidiosis and 2 isosporiasis; 12 mycobacterial enteritis; 15 CMV enteritis; 7 candidosis; 7 intestinal microsporidiosis confirmed by electron microscopic examination; 12 Giardiasis; 3 duodenal leishmaniasis; 1 intestinal cryptococcosis). Multiple concurrent infections were noted in 6 cases. A mild to severe villous atrophy was observed in 28 cases, associated with opportunistic infection. A patchy distribution of pathogen agent was noted in 34 cases, and 37 cases were associated with oesophagal candidosis. This study points out the value of histologic examination of intestinal biopsy for the diagnosis of systemic infections as well as of unusual parasitosis, and the necessity for multiple endoscopic biopsies because of the frequent patchy distribution of pathogens.


[Stromal luteoma of the ovary. Differential diagnosis of steroid cell tumors]. An observation of a stromal luteoma of the ovary is reported. It is a rare tumor (only about thirty cases of it have been published yet), occurring mostly in post menopausal women. Endocrine symptoms and sometimes virilizing signs may be observed. Abnormal vaginal bleeding is the most frequent clinical manifestation. This macroscopically observed tumor is surrounded by ovarian stroma and entirely composed of luteinized cells devoid of crystals of Reinke. Hyperthecosis of ovarian stroma is often observed. Its evolution is always benign. The authors recalled the place of stromal luteoma of the ovary among steroid (lipid) cell tumors and the elements of a differential diagnosis. These tumor might derive from ovarian stromal cells.


Effects of different dosages and modes of sodium bicarbonate administration during cardiopulmonary resuscitation. Systemic acidosis occurs during cardiac arrest and cardiopulmonary resuscitation (CPR). The present study investigated the effect of different modes of sodium bicarbonate administration on blood gas parameters during CPR. Arterial and venous blood gases were obtained during 10 minutes of CPR which was preceded by 3 minutes of unassisted ventricular fibrillation in 36 dogs. Following 1 minute of CPR, the animals received one of four treatments in a randomized and blinded manner: normal saline (NS), sodium bicarbonate bolus dose 1 mEq/kg (B), sodium bicarbonate continuous infusion 0.1 mEq/kg/min (I), and sodium bicarbonate bolus dose (0.5 mEq/kg) plus continuous infusion 0.1 mEq/kg/min (L+I). Eleven dogs completed NS, 8 B, 8 I, and 9 L+I protocol. Following NS infusion, both arterial and venous pH declined consistently over time. Significant differences compared with NS treatment in venous pH were observed at 12 minutes of ventricular fibrillation (L+I, 7.27 +/- 0.05; NS, 7.15 +/- 0.05; B, 7.20 +/- 0.05; I, 7.24 +/- 0.04, each bicarbonate treatment versus NS, and L+I versus B, (P < .05). The B group had an elevated venous PCO2 (mm Hg) concentration following 6 minutes of ventricular fibrillation compared with NS, L+I, and I groups (81 +/- 14 versus 69 +/- 10 versus 68 +/- 10 versus 71 +/- 8, respectively, (P = .07). Arterial pH and PCO2 values showed a similar trend as the venous data with the L+I group demonstrating arterial alkalosis (pH > 7.45) at 12 minutes of ventricular fibrillation.(ABSTRACT TRUNCATED AT 250 WORDS)


The biochemical mechanisms of the excitotoxicity of kainic acid. Free radical formation. Kainic acid (KA) is a known potent neuroexcitotoxin, although the biochemical mechanism producing its underlying neurotoxic effect is not quite clear. Histopathological examination of gerbil brains 24 h after systemic injection of KA revealed severe neuronal lesions in different regions of the brain, especially the cerebellar and hippocampal areas. We have detected free radical formation in the brain 1 h after KA administration by using an in vivo spin trapping technique. We have also observed increased lipid peroxidation in the brain after KA-treatment by analyzing thiobarbituric acid reactive substances and conjugated diene formation. Diminished brain specific (Na+, K+)-ATPase activity was also found 2 h after KA injection and persisted to 24 h. It is possible that the free radical reaction is a primary cause of neuronal degeneration after KA administration.


[The acoustics of the open mastoid cavity (so-called "radical cavity") and its modification by surgical measures. II. Clinical studies]. The acoustic resonance of a severely altered outer ear channel (radical mastoid cavity) is investigated in a series of 18 patients who underwent revision surgery by means of in-situ measurements of the sound-pressure-level near the tympanic membrane. While the average volume of the open cavity differs from the normal ear channel for the factor 2.5, the size of the external meatus is--in average--only 20% larger. This leads to an average frequency in patients with open cavity of 1939 Hz, more than 1000 Hz less than in a series (n = 20) of normal ears (average resonance frequency: 2942 Hz). The altered acoustic behaviour of the open cavity leads to partial extensive discrepancies of the resonance-caused sound-pressure augmentation in the frequencies of 3 and 4 kHz, which are important for speech perception. The average difference is more than 10 dB (SPL). Proved surgical techniques of cavity obliteration and meatoplasty can lead to a nearly normalized acoustic behaviour of the outer ear in a statistic significant way. Due to these surgical procedures, an average postoperative resonance frequency of 2421 Hz could be reached in our patients. Especially, the resonance-caused sound-pressure augmentation in 3-4 kHz could nearly be equalized to such of a normal outer ear. Differences in the acoustic behaviour of the outer ear as can be found between patients with an open mastoid cavity and normal ears can almost be eliminated surgically.(ABSTRACT TRUNCATED AT 250 WORDS)


Acid base changes in arterial and central venous blood during cardiopulmonary resuscitation. Twenty-seven patients in cardiopulmonary arrest had simultaneous measurements of arterial and central venous blood gases during cardiopulmonary resuscitation (CPR) with a pneumatic chest comparison and ventilation device. Mean central venous and arterial hydrogen ion concentrations, PCO2 and calculated bicarbonate concentrations were significantly different (P less than 0.01) at all sampling times (0, 10 and 20 min). Central venous blood samples predominantly showed a respiratory acidosis in contrast to a mixed disturbance in arterial samples inclined towards a metabolic acidosis. The mean difference between central venous PCO2 (pcv CO2) and arterial PCO2 (pa CO2) ranged from 5.18 to 5.83 kPa reflecting the low blood flow in patients undergoing CPR. Measurement of arterial Po2 indicated adequate oxygenation using the pneumatic device. Arterial blood gas analysis alone does not reflect tissue acid base status. Bicarbonate administration during CPR may have adverse effects and any decision as to its use should be based on central venous blood gas estimations.


Studies on the compatibility of diethylpropion hydrochloride with carboxymethylcellulose and other dietary fibres. The combination of diethylpropion hydrochloride with carboxymethylcellulose induces decomposition of the drug. The principal decomposition products obtained were 1-phenyl-1,2-propanedione, benzoic acid and diethylamine as hydrochloride and benzoate salts. These phenomena have been explained with reference to basic decomposition scheme of the diethylpropion. Additionally, the behaviour of diethylpropion in mixtures with other dietary fibres such as methylcellulose, wheat bran and wheat germ was examined. Diethylpropion in combination with methylcellulose is rather stable while in mixtures with wheat bran and wheat germ suffers degradation. An increase in the weight of the excipient, probably due to chemical binding of the drug and other degradation products by the excipient, was observed in all these cases. The decomposition reactions were studied by applying a sequence of selective solvent extractions and using high-performance liquid chromatography (HPLC).


Brainstem auditory evoked responses in infants and children with AIDS. Brainstem auditory evoked responses were measured in 16 infants and children with acquired immunodeficiency syndrome (AIDS) and in 9 normal infants and children. Two stimulation rates were used: a conventional rate of 10 Hz and a high rate of 50 Hz. Latencies of waves III, IV, and V on the left were significantly longer in the AIDS group when a stimulation rate of 10 Hz was used. With a higher stimulation rate of 50 Hz, significant differences between the two groups occurred in the latencies of waves I, III, and V bilaterally, but there were no significant differences in the interpeak latencies. A measure of the differential effect of the increasing stimulus rate on the two groups was significant for wave I latency and for I-III and I-V interpeak latencies on the left, revealing that increasing stimulation rate prolongs these measures more in the AIDS group. Increased brainstem auditory evoked response stimulation rate may unmask abnormalities in infants and children with AIDS that are not observed when the lower stimulation rate is used.


Bombesin receptor antagonists. 5 new irreversible alkylating analogues. New alkylating bombesin analogues were synthesized in order to increase their solubility and stability in aqueous solutions. The best compromise between these parameters and the biological properties (receptor binding and antagonistic activity) was achieved with 4-[bis(2-chloro-ethylamino)]benzoyl derivatives of the BN (7-14) octapeptide carrying a (13-14) reduced peptide bond independently of the presence of a His12 residue, either free or protected.


New 1,2,4-oxadiazole derivatives: synthesis and adrenergic receptors binding studies. In order to obtain derivatives with simultaneous alpha- and beta-adrenergic blocking activity, compounds having the phenoxypropanolaminic structure of beta-adrenergic blockers have been synthesised, as well as 1,2,4-oxadiazole moiety, which could imitate the imidazolinic nucleus characteristic of drugs acting on alpha-adrenergic receptors. The synthesised compounds have been submitted to alpha and beta receptor binding assays. Some derivatives showed an alpha-adrenoceptor binding activity higher than labetalol and similar to prazosin, but with a poor beta-adrenoceptor binding activity.


[Several immunological characteristics of occupational exposure to asbestos and other mineral fibers]. The authors evaluated some immunologic factors in workers exposed to asbestos, basalt and glass fibres and compared the results with a control reference group. The findings indicate that there is a considerable difference between the immunologic reaction of the organism, mainly when the humoral component of the answer is taken into account, in the exposed and in the control group. The same observation was valid, when the asbestos exposed group was compared with the groups exposed to asbestos substitutes (basalt and glass fibres). The immunoglobulins of the classes A, G, M, complement components C3 and C4 were significantly increased, alpha-1-antitrypsin and phagocytic activity of leukocytes in peripheral blood was decreased. Most of the examined indicators in the basalt- and glass fibres exposed groups differed insignificantly from the control group. Transferrin level was not significantly different in any of the examined groups. The results indicate that there is higher tolerability of basalt and glass fibres in the organism compared with asbestos fibres.


Opioid and non-opioid effects of novel butyrophenone analogues. Haloperidol, haloperidol propionate, and a haloperidol analogue N-3-(p-fluorobenzoyl) propyl-4-phenyl-4-propionyl-oxypiperidine (NIH 10495) were evaluated in several in vitro and in vivo tests of opioid effects. Haloperidol bound to opioid receptors with very low affinity and had no opioid agonist effects in the other test systems. Haloperidol propionate was 10 times less potent than NIH 10495 in the binding assay and in the smooth-muscle assay. Both of these haloperidol analogues decreased the rate and volume of respiration in air and in 5% CO2 with NIH 10495 being approximately 50 times more potent than haloperidol propionate. The NIH 10495, but not the haloperidol propionate, attenuated naltrexone-like discriminative stimulus effects in morphine-dependent withdrawn rhesus monkeys. Intravenously delivered NIH 10495 maintained higher rates of responding than did haloperidol propionate when evaluated for reinforcing effects. These drugs appear to have novel spectra of action that suggest possible value for this synthetic approach to the development of clinically useful analgesics and to the development of novel neuroleptics.


Comparison of the effects of remoxipride and raclopride on nigrostriatal and mesolimbic dopaminergic neuronal activity and on the secretion of prolactin and alpha-melanocyte-stimulating hormone. Raclopride and remoxipride, which are reported to be selective dopamine-D2 antagonists, are currently under clinical investigation as antipsychotic drugs. The present study compared the relative abilities of these two drugs to alter the activity of nigrostriatal and mesolimbic dopaminergic neurons, and plasma levels of hormones originating from the anterior and intermediate lobes of the pituitary in male rats. Although raclopride was consistently more potent than remoxipride, both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid in the striatum and nucleus accumbens, which contain terminals of nigrostriatal and mesolimbic dopaminergic neurons, respectively. Both drugs also caused significant dose- and time-related increases in plasma levels of prolactin, but only raclopride increased plasma levels of alpha-melanocyte-stimulating hormone (alpha-MSH). These results suggest that although raclopride and remoxipride are both classified as D2 receptor antagonists they can be distinguished from one another by their relative ability to block the inhibitory dopaminergic control of alpha-MSH from melanotrophs in the intermediate lobe of the rat pituitary.


The effects of ethanol on human sleep EEG power spectra differ from those of benzodiazepine receptor agonists. A single dose of ethanol (0.60 g/kg of body weight) was administered to eight young healthy male subjects 35 minutes before bedtime. Compared to the average value of two baseline nights, subjective sleep and polysomnographically determined sleep parameters were not significantly affected. In the first 2 hours of sleep after ethanol intake, the combined value of wakefulness, stage 1, and movement time was reduced. In this interval, visually scored stage 4 sleep was increased, and electroencephalographic (EEG) power density in nonrapid-eye-movement (nonREM) sleep was enhanced in the lowest delta frequencies and reduced in the beta range. Computed for the entire sleep episode, power density in REM sleep was enhanced in some theta frequencies. In the sleep episode initiated 24 hours after ethanol intake, power density in nonREM and REM sleep was enhanced in delta and theta frequencies, and the subjectively perceived number of awakenings was reduced. The effects of ethanol on EEG power spectra during sleep differ from those published for benzodiazepine and nonbenzodiazepine hypnotics. This indicates that the effects of ethanol on the human sleep EEG are not mediated by the benzodiazepine receptor.