[Effect of light on total micro-bilirubin values in vitro]. In order to elucidate the effect of light on total bilirubin values in vitro, 616 capillary blood samples were collected from jaundiced newborn infants at the nursery of Veterans General Hospital-Taipei. Samples were divided into 3 groups: 1. Phototherapy light group-133 samples were irradiated with blue fluorescent light (spectral irradiance 425-475 nm = 4 mu watt/cm2/nm); 2. Room light group-202 samples were irradiated with white fluorescent light (spectral irradiance 425-475 nm = 0.2 mu watt/cm2/nm); 3. Dark group-the last 215 samples were placed in the dark. Total bilirubin values were checked with spectrophotometry, at 0, 2, 4, 6, 24 and 48 hours after being placed in different environments. Total bilirubin values varied significantly in different light source and time groups (p less than 0.0001), plus both had statistical interactions (p less than 0.0001). There were significant decreases in the bilirubin values of the phototherapy group beginning at 2 hours (p less than 0.05), and in the room light group beginning at 6 hours (p less than 0.05), but there was no change in the dark group. We conclude that blood samples for total bilirubin values should not be placed under the light of phototherapy even 2 hours. If blood samples are exposed to room light inevitably, it is safe to be checked within 4 hours. If immediate measurements are unavailable, the samples can be placed in a dark environment allowing the values to remain unchanged for 48 hours.


[A comparison of transrectal fine needle aspiration and biopsy of prostatic lesion]. Transrectal fine needle aspiration and biopsy were performed on 100 patients with suspicious prostatic lesions using 22 gauge aspiration needles and 18 gauge biopsy needles. These collections were made from January 1990 to June 1991. Sufficient prostate tissue for cytological and histological diagnosis were obtained in 97% (97/100) and 99% (99/100), respectively. Thirty-nine patients (39%) were diagnosed having cancer with an accuracy rate of 89.7% (35/39) by histology and 69.2% (27/39) by cytology. One patient with a normal cytological aspiration was found to have an atypical biopsy, therefore, the biopsy was repeated and the prostate showed adenocarcinoma. Our results indicate that the needle aspiration cytology has a less accurate diagnosis rate than the needle biopsy histology. Fine needle aspiration is a safe and effective outpatient procedure but there is a definite learning curve before its use can be fully exploited. It is important to have experienced urologists and pathologists to perform these aspirations until sufficient experience is accumulated.


[The incidence and cause of death following surgery for benign biliary tract disease]. A total of 4056 consecutive patients operated for benign biliary tract disease between January 1983 and June 1990 were reviewed retrospectively. There were 71 in-hospital deaths, representing 1.75% of morality rate. Cholecystectomy was performed in 2275 patients; common bile duct exploration was carried out in 1494, with operative mortality rates of 0.4% and 3.1% respectively. Sepsis was the leading cause of mortality. The second common cause of death was hepatic failure, and respiratory failure the third. Cardiovascular problems accounted for 11.3% of death, which was much lower than reports from western series. Other causes of mortality included hypovolemic shock, anaphylactic shock, epilepsy and hypoglycemia. Of the total 4056 patients, 28.5% underwent emergency surgery. The mortality rate for emergency surgery was 3.6 times higher than for those who underwent elective operations (p less than 0.005) Significantly higher mortality rates were also seen for those aged over 70 years and of male gender. Because the mortality rate of patients who underwent emergency operation due to acute inflammatory or obstructive complications were much higher than that of elective operation group, we suggested early surgical intervention for the symptomatic patients before complications occurred.


[Radiotherapy of the maxillary sinus cancers]. Between 1983 and 1988, 23 patients with cancer of maxillary sinus were treated with either postoperative radiotherapy or radiation alone at the Radiation Oncology Department, Veterans General Hospital-Taichung. There were 20 males and 3 females, 38 to 80 years of age with median age of 60 years. Nineteen were of epidermoid type and 4 were of other histologic types. There were 11 cases of T3 and 12 cases of T4. Only 1 case had neck lymph node metastasis. All patients had bony destruction shown in sinus X-ray films before treatment. Four patients received radiation alone because they refused or were unsuitable for operation. Nineteen patients received postoperative radiotherapy. For radiation alone 70-80 Gy in 7-8 weeks was given in open fields or wedge filters by cobalt-60 gamma radiation. For postoperative radiotherapy, all patients were treated with wedge filters with 50-80 Gy in 5-8 weeks by cobalt-60 radiation also. The minimum followup period was 29 months. All patients treated with radiation alone expired within 2 years with median survival time of 9 months. The 5-year survival rate of postoperative therapy was 40.3% with median survival time of 26.8 months. The overall 5-year survival rate was 33.4% with median survival time of 17 months. There was no significant difference in 5-year survival rate between T3 (34.1%) and T4 (33.3%). The data indicated no survival difference in different types of operation. The therapy of radiation alone was not advocated and the total dose of postoperative radiotherapy should be over 60 Gy for curative purpose.


Idiopathic portal hypertension: a case report. Clinically, idiopathic portal hypertension (IPH) is characterized by overt splenomegaly with pancytopenia, portal hypertension and relatively mild abnormalities in liver function tests. Although its etiology is still undetermined, the liver pathology is characterized by occlusive changes of the intrahepatic portal radicles, portal and periportal fibrosis, irregularly distributed parenchyma atrophies and absent of regeneration nodules. The disease is relatively benign and does not progress to cirrhosis. Differential diagnosis between IPH and liver cirrhosis is mandatory. We now report a case with histologically proven IPH, including clinical course, laboratory data, roentgenographic findings of hepatic venogram and celiac angiogram, hepatic hemodynamic features and intravariceal pressure of esophageal varix which has never been reported in Taiwan.


Persistent interictal aphasia after repeated partial seizures: a case report. We reported a case of persistent interictal aphasia after repeated partial seizures. This 64-year-old man, left-handed and literate, developed repeated right facial twitching associated with head turning to the right or transient loss of awareness since October 30 1991. Persistent aphasia between seizures occurred from November 4. An electroencephalogram (EEG) on November 6 showed an epileptic focus over the left frontal area. Tc-99m HMPAO brain single photon emission computed tomography (SPECT) performed one and half and hour after an attack on November 7 showed focal hyperperfusion over the left frontal area. The aphasia recovered on November 7 after the seizures had been stopped. Follow-up EEG and Tc-99m HMPAO brain SPECT done on November 11 and November 14 respectively were normal.


Evaluation of carcinoembryonic antigen, tissue polypeptide antigen, and squamous cell carcinoma antigen in the detection of cervical cancers. Cervical cancer is a predomiannt gynecologic neoplasia in Taiwan. The associations of carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA) and squamous cell carcinoma antigen (SCC-Ag) for detection of cervical cancers with type of cancer, clinical stage and lymph node metastasis were studied. Serum samples were collected from 395 patients with cervical cancers (61 CIN3, 334 squamous cell carcinoma, 18 adenocarcinoma), 72 patients with benign gynecologic diseases and 115 healthy controls. The sensitivities of CEA, TPA, and SCC-Ag were 15.2%, 33.6%, and 49.3%. The specificities were 89.3%, 96.2%, and 95.1%, respectively. The positive rate of CEA was found significantly higher among patients with adenocarcinoma than those with squamous cell carcinoma. The positive rate of CEA and SCC-Ag increased with advance of the clinical stages. There was no significant difference in the rate of each tumor marker between patients with lymph node metastasis and those without metastasis. The positive rate obtained by SCC-Ag alone was comparable to those by the combinations of two or three antigens. The result of the present study indicates that SCC-Ag is the most useful tumor marker in the detection of cervical cancers.


Pure rhabdomyosarcoma of the corpus uteri in a postpartum patient: report of a case and review of the literature. Pure rhabdomyosarcomas originate in the female genital tract. They are uncommon and most often occur in infancy or childhood as sarcoma botryoides (embryonal rhabdomyosarcoma) which involve the vagina and cervix. Such tumors rarely occur in adults. A pure rhabdomyosarcoma of the uterus that arose in a postpartum patient is described. The pertinent literature is discussed.


Chronic progressive external ophthalmoplegia with NADH-CoQ reductase deficiency: report of a case. A thirty-two year old female had chronic progressive external ophthalmoplegia (CPEO), exertional fatigue, dysarthria, dysphagia, and bilateral hearing impairment. Histochemical stains, obtained from the right vastus lateralis, showed ragged-red fibers and wide-spread abnormalities in the number, size, and the structure of mitochondria under electronomicroscopic examination. A biochemical analysis showed a low activity of NADH-cytochrome C reductase, NADH dehydrogenase and a normal activity of succinate cytochrome C reductase and cytochrome C oxidase. This data suggests a specific defect in the NADH dehydrogenase of complex I (NADH CoQ reductase). We believe that this is the first biochemically defined mitochondrial myopathy reported in Taiwan and provides additional evidence for the existence of biochemical heterogeneity in mitochondrial disorders of CPEO.


DNA image cytometry in stomach carcinoma. Its relation to histomorphologic parameters and its influence on prognosis. The influence of DNA content on the prognosis in stomach cancer is controversial. After curative resection of stomach carcinomas (adenocarcinomas [n = 58]; signet ring cell carcinomas [n = 24]; undifferentiated carcinomas [n = 21]), the influence of the DNA content and histomorphologic parameters on the prognosis was examined. In the multivariate regression analysis, the prognosis depended on the lymph node status (P = 0.0009), pT stage (P = 0.02), tumor localization (P = 0.03), and histologic type (P = 0.05). The prognosis was independent of the DNA content. Furthermore, the degree of differentiation, operative procedure, safety distance, size of the tumor, and sex and age of the patient also did not influence the prognosis. The DNA content of the tumor cells in stomach carcinoma does not influence prognosis.


Histologic follow-up of ampullary adenomas in patients with familial adenomatosis coli. The major duodenal papilla is a common site of extracolonic adenoma in patients with familial adenomatosis coli (FAC). However, there have been no reports which have systematically discussed histologic change in atypia of ampullary adenomas with time by their location in the papilla. The major duodenal papillae of 23 patients with FAC were followed endoscopically and histologically for an average of 7.7 years (range, 1 year to 14 years 7 months). Tubular adenomas were detected histologically in 17 of the 23 patients at the first (14) or following examinations (3). They occurred in the orifice and/or ampulla in 11 patients and in the surface of the papilla in 8 patients. Three of the 11 orifice and/or ampulla adenomas contained moderate to severe atypia. There was no histologic change in atypia or malignant transformation during the follow-up period. In the patients with FAC, the major duodenal papilla had adenoma, i.e., precancerous lesion, at a high incidence (74%), and it is reported that the ampulla of the papilla tended to have extracolonic carcinoma. Therefore, it is necessary to follow duodenal papillae of patients with FAC carefully and take biopsy specimens repeatedly from various sites, especially from the orifice or ampulla even if the papilla seems to be normal.


Continuous-infusion cisplatin, 5-fluorouracil, and bolus methotrexate in the treatment of advanced non-small cell lung cancer. Cisplatin and 5-fluorouracil have noted synergy in preclinical systems. The authors combined methotrexate with infusional cisplatin and 5-fluorouracil in an attempt to produce a regimen with improved activity in advanced NSCLC: Twenty-six ambulatory patients with previously untreated non-small cell lung cancer were treated with continuous-infusion cisplatin (25 mg/m2/day for 5 days), 5-fluorouracil (800 mg/m2/day for 5 days), and intermediate-dose methotrexate (200 mg/m2 on days 15, 22), followed by leucovorin rescue (PFM regimen). Patients received a median of four cycles of therapy. Two patients had a complete response, and 10 had a partial response (overall response rate, 46.2% or 12 of 26). The median time to treatment failure was 22.5 weeks; the median survival was 55 weeks from the start of chemotherapy. There were no toxic deaths attributed to chemotherapy. Thrombocytopenia was the only Grade 4 toxicity (27%). Grade 1/4 and 2/4 peripheral neuropathy occurred in 17 of 26 patients (66%) and was associated with a cumulative cisplatin dose of more than 300 mg/m2. PFM (using continuous-infusion cisplatin) produced a high response rate but resulted in an high incidence of low-grade peripheral neuropathy.


Low molecular weight heparin for Hickman catheter--induced thrombosis in thrombocytopenic patients undergoing bone marrow transplantation. Patients with an indwelling central venous catheter are prone to development of thrombotic complications. Thrombocytopenia in patients undergoing bone marrow transplantation (BMT) generally is protracted. The management of thrombosis in thrombocytopenic patients is difficult because heparin and warfarin are relatively contraindicated because of the high risk of major bleeding. Low molecular weight heparin (LMWH) is a new class of antithrombotic drug. Enoxaparin (Rhone Poulenc Rorer, Antony, France) is an LMWH that has been shown to be effective in the treatment and prophylaxis of venous thrombosis. Enoxaparin, with its high antithrombotic to anticoagulant ratio, may be safer than standard heparin in patients at high risk of hemorrhagic complications. The authors report the cases of five thrombocytopenic patients, undergoing autologous BMT, in whom venous thrombosis developed related to a Hickman catheter. All the patients were treated with Enoxaparin and recovered promptly without hemorrhagic complications. The authors suggest that Enoxaparin is an effective drug in treating thrombocytopenic patients in whom acute venous thrombosis develops.


High frequency of human papillomavirus infection in carcinoma of the urinary bladder. The prevalence of type 6, 11, 16, 18, and 33 human papillomavirus (HPV) was investigated with the polymerase chain reaction (PCR) on formalin-fixed, paraffin wax-embedded material, including 48 neoplastic and 21 normal urinary bladder specimens. The PCR-amplified DNA were analyzed by gel electrophoresis and dot blot and Southern blot hybridization. Some tissues were tested further by nonisotopic in situ hybridization. HPV DNA was detected in 39 (81%) of 48 carcinomas and 7 (33%) of 21 normal urinary bladder specimens. The presence of high-risk HPV (types 16, 18, and 33) was increased significantly in carcinoma cases (62%) as compared with normal specimens (14%) (P less than 0.01). Similarly, multiple HPV infections were significantly higher in carcinoma (60%) than in the normal tissues (5%) (P less than 0.01). The overall and high-risk HPV infections in both neoplastic and normal specimens were distributed almost equally in male and female patients. There was no significant correlation between positive results for HPV and histologic grades of the carcinoma. These results demonstrate that the urinary bladder in both sexes is another site where infection with the common genital tract HPV may carry a risk of malignant transformation.


From serotonin receptor classification to the antimigraine drug sumatriptan. After the synthetic serotonin 5-hydroxytryptamine (5-HT) became available in the early 1950s, attempts were soon under way to study the nature of 5-HT receptors. Using the guinea-pig isolated ileum, Gaddum and Picarelli (1957) suggested that 5-HT-induced contractions were mediated by a morphine-sensitive "M" receptor located on the parasympathetic ganglion and a dibenzyline-sensitive "D" receptor located on the smooth muscle. Though this classification ws used during the next three decades, it was realized that some effects of serotonin, for example vasoconstriction within the carotid vascular bed, were not mediated by either "M" or "D" receptors. When radioligand binding studies led to the identification of 5-HT1 and 5-HT2 "receptors" in the rat brain membranes, it became increasingly apparent that the two receptor classifications were not identical. Thus, a new framework for serotonin receptor nomenclature and classification was proposed: 5-HT1-like (5-HT1), 5-HT2 (formerly "D") and 5-HT3 (formerly "M") receptors. At the present time, several subtypes of 5-HT1 receptors as well as a 5-HT4 receptor are also recognized. As the serotonin receptor classification was emerging to indicate that carotid vasoconstriction by serotonin is mediated by a subtype of 5-HT1 receptors, on the migraine front it was being suggested that the disease is associated with vasodilation within the cranial extracerebral circulation and deranged serotonin metabolism and that certain antimigraine drugs caused a selective carotid vasoconstriction, probably via serotonin receptors. Therefore, Humphrey and colleagues conceived that synthesis of serotonin derivatives may lead to a compound that would elicit highly selective carotid vasoconstriction and abort migraine attacks. Indeed, via the synthesis of 5-carboxamidotryptamine and AH25086, sumatriptan was designed. The drug acts as an agonist at the vasoconstrictor 5-HT1 receptor subtype and has proved highly effective in the therapy of migraine attacks.


Sumatriptan is a potent vasoconstrictor of human dural arteries via a 5-HT1-like receptor. The action of sumatriptan, putatively a selective 5-HT1D or 5-HT1-like receptor agonist which is effective in the treatment of migraine, has been studied on fresh human dural (middle meningeal) arteries. In low concentrations (10(-8)-10(-7) M) it was found to be a significantly stronger vasoconstrictor of dural arteries compared to cerebral and temporal arteries. However, its potency was less than that of 5-HT. The sumatriptan-induced vasoconstriction was antagonized by methiothepin (10(-9)-10(-8) M), but not by ketanserin (10(-7) M). The observations suggest that the sumatriptan-induced contraction of the dural artery is mediated via activation of 5-HT1D or 5-HT1-like receptors, whereas it does not appear to activate the 5-HT2 receptors.


Comparative effects of the antimigraine drugs sumatriptan and ergotamine on the distribution of cardiac output in anaesthetized pigs. The haemodynamic effects of sumatriptan, a 5-HT1-like receptor agonist, and ergotamine, an agonist at alpha-adrenergic, dopamine as well as 5-HT receptors, were compared using intracardiac injection of radioactive microspheres of different sizes in anaesthetized pigs. Ergotamine (0.02 mg.kg-1) and sumatriptan (0.3 mg.kg-1) decreased systemic vascular conductance and cardiac output. Only ergotamine raised arterial blood pressure. Both sumatriptan and ergotamine decreased arteriovenous anastomotic, but not capillary, blood flow in the head and body skin. Arteriovenous and capillary blood flow in the dura mater and nasal mucosa and capillary blood flow in the brain, kidneys, adrenals, intestine, heart, spleen and muscle remained unchanged. However, kidney conductance was decreased by both drugs, spleen conductance by sumatriptan and heart, liver and adrenal conductances were decreased by ergotamine. Thus, both sumatriptan and ergotamine constricted arteriovenous anastomoses in the skin, but not in the dura mater or nasal mucosa. Ergotamine constricted the vasculature more than sumatriptan, although both drugs may differentially decrease vascular conductances in some organs.


Sumatriptan injection is superior to placebo in the acute treatment of migraine--with regard to both efficacy and general well-being. The efficacy of subcutaneous injection of sumatriptan in the acute treatment of migraine was assessed in a double-blind, randomized, placebo-controlled cross-over study of 27 migraine patients. In addition, the patients were asked to give information about their well-being and subjective symptoms by means of a self-administered standardized questionnaire. A total of 22 migraine sufferers received a subcutaneous (sc) injection of 8 mg of sumatriptan and 24 received placebo. Of these patients, 19 received both treatments and thus completed the study. The primary efficacy end-point was a reduction in headache severity from severe or moderate to mild or no headache at 30, 60, 90 and 120 min. An effective response to treatment was achieved within 30 min in 63% and within 60 min in 84% of patients when treated with 8 mg sumatriptan sc, compared with 11% for placebo (p less than 0.001). Sumatriptan also provided significant relief from nausea and photophobia as compared with placebo. The proportion of patients that needed rescue medication after 120 min was significantly lower (p less than 0.001) with active treatment when compared with placebo. Sumatriptan was well tolerated and the majority of adverse events were mild and transient. The most frequent symptoms were those of malaise/fatigue or numbness. No changes in blood pressure or ECG readings were observed during the treatment. Compared with placebo, subcutaneous 8 mg sumatriptan also caused a substantial improvement in general well-being as revealed by the Minor Symptoms Evaluation Profile-acute (MSEP-acute) questionnaire.(ABSTRACT TRUNCATED AT 250 WORDS)


Sumatriptan in chronic cluster headache: results of continuous treatment for eleven months. A 32-year-old man received a total of 480 injections of subcutaneous sumatriptan 6 mg for the treatment of acute attacks of chronic cluster headache over an eleven-month period. Over 90% of the attacks resolved within 10 min of treatment (average 6.8 +/- 3.4). As a comparison, the average duration of 61 attacks occurring over the same period, but not treated with sumatriptan, was 56.1 +/- 20.8 min. This difference was highly significant. There was no clinical evidence of tachyphylaxis, and there were no adverse effects. This is the first report of a long-term treatment of cluster headache with sumatriptan. It is concluded that sumatriptan in this case was an effective and well-tolerated treatment for cluster headache.


Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors. Using several naturally occurring and synthetic retinoic acid (RA)-responsive reporter genes, we show that the patterns of transcriptional activation by various retinoic acid receptor (RAR) and retinoid X receptor (RXR) forms vary according to the nature of the RA response element and the context of the stimulated promoter. We demonstrate the presence of autonomous, ligand-inducible, and promoter context-dependent transactivation functions (AF-2s) located in the C-terminal region of all RARs and RXRs. In addition, promoter context-specific modulating transactivation functions are associated with the N-terminal A and B regions of RARs and RXRs. We also show that these transactivation and modulating functions exhibit response-element specificity. The modulating functions display a marked specificity in their cooperation with the AF-2 transactivation functions, cooperation that depends on the receptor origin of the modulating and transactivation functions and the promoter context of the RA-responsive gene, thus accounting for the specific transactivation properties of RAR and RXR types.


The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.


Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. We examined the binding interactions of the methylation-dependent chemotaxis receptors Tsr and Tar with the chemotaxis-specific protein kinase CheA and the coupling factor CheW. Receptor directly bound CheW, but receptor-CheA binding was dependent upon the presence of CheW. These observations in combination with our previous identification of a CheW-CheA complex suggest that CheW physically links the kinase to the receptor. The ternary complex of receptor, CheW, and CheA is both kinetically and thermodynamically stable at physiological concentrations. Stability is not significantly altered by changes associated with attractant or repellent binding to the receptor. Such binding greatly modulates the kinase activity of CheA. Our results demonstrate that modulation of the kinase activity does not require association-dissociation of the ternary complex. This suggests that the receptor signal is transduced through conformational changes in the ternary complex rather than through changes in the association of the kinase CheA with receptor and/or CheW.


Ion transport in colon cancer cell cultures studied by X-ray microanalysis. Three colon cancer cell lines (Colo 205, HT29 and T84) were investigated by X-ray microanalysis with respect to elemental composition and the effect of cAMP on the cellular concentrations of Na, K, and Cl. The cultures were not homogeneous with respect to their elemental composition, but appeared to consist of two sub-groups, low-K cells and high-K cells. In all three cell lines, the low-K cells had, in addition, higher Ca, markedly lower Cl, and somewhat lower P and S concentrations. Differences in Na and Mg concentrations were absent or not consistent. Exposure of cells to cAMP caused a decrease of the cellular Cl and K content in high-K (high-Cl) cells. Changes in Na were not significant. No difference between the three cell lines could be noted. Incubation of the cells with phorbol myristate acetate (PMA), which has been shown to down-regulate the expression of the cystic fibrosis (CF) transmembrane conductance regulator gene and thus confer CF-like characteristics on the cells, significantly decreased the response in the cellular Cl concentration to cAMP stimulation. It is concluded that cAMP initially activates predominantly the apical Cl- channel and the basolateral K+ channel.


Expression of SV40 T-antigen in lipoma cells with a chromosomal translocation T(3;12) is not sufficient for direct immortalization. 12q13-15 changes are the most frequent cytogenetic abnormalities in human tumor cells. To test their biological significance we used an assay based on lipoma cells with a limited in vitro lifetime and this type of chromosomal aberration. Lipoma cells with a reciprocal translocation t(3;12)(q28;q14) were transformed by transfection with a plasmid containing the SV40 "early region". The transformed cells showed an altered morphology with loss of contact inhibition, formation of foci, and T-antigen expression. They were immortalized after a growth crisis. The karyotypic patterns before and after the crisis show that the translocation together with expression of SV40 T-antigen is not sufficient for direct immortalization.


Cytokine abnormalities in inflammatory arthritis. Cytokines are soluble molecules which control communication between cells of the immune and non-immune systems. Studies on their role in the pathogenesis of inflammatory arthritis have been increased with the discovery of new cytokines and the development of assays for their detection. Conditions such as rheumatoid arthritis are characterized by increased production of proinflammatory cytokines in association with reduced control by regulatory cytokines produced by T lymphocytes. The inadequate inhibition of proinflammatory cytokines by anti-inflammatory cytokines and other regulatory mechanisms contributes to this cytokine imbalance. This situation is responsible for the enhanced degradation, without sufficient repair activity. These results have provided the rationale for the use of cytokines as well as for drug targeting of the cytokine network in rheumatoid arthritis. This also includes the modulation of the cytokine network by targeting the level of the receptors as well as the effects and/or the responding cells.


The role of cytokines and their inhibitors in arthritis. The role of cytokines in inflammatory joint diseases is well documented, especially with regard to tissue destruction and remodelling. In these processes, IL-1 and TNF alpha play a prominent part by stimulating protease production. The regulation of their production, their release and their effects on target cells (e.g. synovial cells, chondrocytes and bone-derived cells) has therefore been the subject of intensive investigations. In this context a new dimension has emerged recently due to the observation of the existence of natural specific cytokine inhibitors. IL-1-ra and the soluble fragments of both TNF receptors--inhibitory by binding to TNF alpha--are natural products. These appear to be the molecules best suited for controlling the imbalance between pro- and anti-inflammatory processes. The use of the recombinant forms of these inhibitors may open new perspectives for therapeutic intervention. The fact that the respective mechanisms of action of receptor antagonists and inhibitory binding proteins differ does not rule out their complementarity. Preliminary experiments with animal models have yielded promising results which should be followed up by clinical trials.


Genetic analysis of serine biosynthesis and glucose repression in yeast. Serine and glycine biosynthesis in yeast proceed by two pathways: a "glycolytic" pathway, using 3-phosphoglycerate, and a "gluconeogenic" pathway, using glyoxylate. We used a mutation in the cat1 gene to abolish the glucose-repressible "gluconeogenic" pathway and re-isolated two mutants, ser1 and ser2, in the "glycolytic" pathway. The ser1 mutation corresponded to phosphoserine transaminase and ser2 to that of phosphoserine phosphatase. Mutagenesis of a ser1 ser2 cat1 triple mutant facilitated the isolation of a mutation in a new gene, SER10. SER10 appears to be part of a pathway which, under normal growth conditions, is less important in serine biosynthesis. The ser1 ser2 ser10 triple mutants were totally serine auxotrophic on glucose media but serine prototrophic during growth on non-fermentable carbon sources. This phenotype was used to select for possible regulatory mutants that synthesize serine by the gluconeogenic pathway even in the presence of glucose, e.g., with a non-glucose repressible glyoxylate cycle. In an alternative approach to isolate such mutants URA3 and TRP1 expression were placed under the control of the glucose-repressible FBP1 (fructose-1,6-bisphosphatase) promoter. Although both systems resulted in strong selection pressure we could not isolate constitutively derepressed mutants. These results indicate that transcription of glucose-repressible gluconeogenic enzymes is mainly dependent on positive regulatory elements.


The CCS1 gene from Saccharomyces cerevisiae which is involved in mitochondrial functions is identified as IRA2 an attenuator of RAS1 and RAS2 gene products. The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation. Homologous integration of this DNA in the genome occurs at the CCS1 locus. An 11 kb of the DNA insert is necessary for complementation. Sequencing part of the fragment identifies CCS1 as the IRA2 gene. The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins. This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function. We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.


Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.


Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit I and II genes, the ATPase9 gene, the NADH dehydrogenase ND4L and ND5 gene complex, and the glutaminyl, methionyl and arginyl tRNA genes from Trichophyton rubrum. In this paper, we present the nucleotide sequence of a 5248 bp-long region of the mitochondrial (mt) genome of the dermatophyte Trichophyton rubrum. This region which represents about 1/4 of the total mt genome of this species reveals a compact organization of genes including: the glutaminyl tRNA, the methionyl tRNA, the cytochrome oxidase subunit I gene, the arginyl tRNA, the mitochondrial version of the ATPase subunit 9 gene, the cytochrome oxidase subunit II gene and a part of the NADH dehydrogenase ND4L and ND5 gene "complex". The main features of the part of mt DNA sequenced is the non-interrupted COXI gene and the presence in the mitochondrial version of the ATPase 9 gene of a small group IA intron. The extensive amino-acid sequence similarity with the equivalent gene in Aspergillus nidulans and Neuropora crassa indicates that this gene codes for a dicyclohexylcarbodiimide binding protein. The conserved arrangement of this portion of the mt genome and the presence of tRNAs between the protein-coding genes are compatible with a large polycistronic transcript processed by the excision of tRNAs, or similar secondary structures, as proposed for other fungal or mammalian mt DNAS.


[Extraocular changes in congenital aniridia]. The authors draw attention to possible extraocular changes in children with congenital aniridia. Of 31 investigated patients they were found in 5 children. Among these changes, because of its serious character, Wilms tumour holds the first place; it was found by the authors in two children. To ensure its early detection, the authors emphasize the necessity to dispensarize all children with congenital aniridia. Collaboration with an experienced X-ray specialist and child oncologist is essential. In addition to Wilms' tumour congenital aniridia can be associated with serious somatic developmental changes. The authors observed in one child and AGR triad and in two patients deformities of the skeleton of the head and lower extremities.


The chemical synthesis of N-[1-(2-naphthol)]-phosphatidylethanolamine, a fluorescent phospholipid for excited-state proton transfer studies. A procedure for the preparation of N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE) has been developed. The synthesis is based on the Schiff base formation between the NH2 of the phospholipid and the aldehyde moiety of 2-hydroxy-1-naphthaldehyde. Then selective reduction of the imine is used to obtain the stable secondary amine, NAPH-PE. Formation of the intermediate Schiff base and the final product is confirmed by 13C- and 1H-NMR. Similar to free 2-naphthol, the excited-state pKa (pKa*) of its phospholipid derivative appears to be significantly lower than the ground-state pKa. At pH 7.4, the excitation spectrum of NAPH-PE shows no deprotonated species in the ground-state, while the emission spectrum presents a significant contribution of this species. Thus the fluorescent phospholipid exhibits the typical behavior of excited-state proton-transfer probes. NAPH-PE is found to incorporate in dimyristoyllecithin (DML) vesicles. The emission spectrum of the probe inserted in the liposomes is affected by acetate used as a proton acceptor. These properties should also be manifest in other lipid bilayers (e.g., plasma membranes of cells) and used for excited-state proton transfer studies.


Superoxide anion production and intracellular free calcium levels in resting and stimulated polymorphonuclear leukocytes obtained from healthy and arteriosclerotic subjects of various ages. It has been established that phagocytic cells are integral components of advanced arteriosclerotic plaques but their role in plaque formation remains unclear. Therefore, toxic agents, such as superoxide anion produced by polymorphonuclear leukocytes (PMNLs) were studied in a clinically defined group of arteriosclerotic patients suffering from obliterative arteriosclerosis of the lower legs. Owing to a close correlation between O2- generation and calcium, the intracellular free calcium concentrations of PMNLs were measured in a resting state and after stimulation with various agents, for example, opsonized zymosan (OZ), the chemotactic peptide f-met-leu-phe (FMLP), and the calcium ionophore A23187. Healthy aged-matched controls were employed. The patients were divided into two age groups: 30-59 years and 60-80 years. We found that in the younger group of arteriosclerotic patients, superoxide anion production and intracellular free calcium concentrations were increased even in the resting state, and only a slight increase was observed after stimulation compared with healthy controls. Granulocyte responses seemed to be similar, independent of the patient's age, to those found in healthy elderly subjects. Arteriosclerosis appears to be associated with an early aging process expressing marked alterations that are greater than those associated with normal aging.


Development of a proton nuclear magnetic resonance spectroscopic method for determining plasma lipoprotein concentrations and subspecies distributions from a single, rapid measurement. We are developing a method for quantifying plasma lipoproteins by proton nuclear magnetic resonance (NMR) spectroscopy that offers advantages over existing clinical methods. We showed that the major lipoproteins have distinct NMR properties sufficient to permit their concentrations to be extracted from a computer lineshape analysis of the plasma lipid methyl resonance envelope (Clin Chem 1991; 37:377-86). We have now discovered that the spectra of the subspecies within each lipoprotein class are different enough to influence the composite spectrum of that class and hence the spectrum of whole plasma. By including spectra representative of these subspecies as additional components in the lineshape-fitting analysis, a complete concentration profile of very-low-density, low-density (LDL), and high-density (HDL) lipoproteins plus the subspecies distributions within these classes can be simultaneously generated. A pilot study of 30 plasma samples of widely varied composition demonstrated good agreement between NMR-derived values and lipoprotein lipid concentrations and LDL and HDL subspecies distributions determined by gradient-gel electrophoresis.


Low ratio of androstenedione to testosterone in plasma and saliva of hirsute women. To gain insight into the passage of androstenedione (A4) through the salivary gland, we measured concentrations of plasma total (TA4), free (FA4), and salivary (SA4) androstenedione during administration of dexamethasone and synthetic corticotropin to control subjects and hirsute women and compared these data with plasma total (TT), free (FT), and salivary testosterone (ST) concentrations. TA4 and FA4 were significantly lower in control subjects throughout (0, 15, 45, and 75 min) (P less than 0.05). SA4 in control subjects was significantly lower only in the follicular phase at 0 and 15 min. There was no significant difference between the increments in SA4 in response to corticotropin. The concentration of SA4 in the control women at 0 min was not significantly different from the concentration of FA4. At 45 and 75 min after corticotropin administration, however, SA4 was slightly higher than FA4 (P less than 0.01). In hirsute women, however, the concentration of SA4 was significantly lower than FA4 at all times (P less than 0.05). TT, FT, and ST concentrations were about twofold higher in the hirsute women than in control subjects throughout. In both groups, ST concentrations were three times as high as FT concentrations (P less than 0.001). The SA4:ST ratio was significantly lower than the FA4:FT ratio in both groups (P less than 0.001) because of higher ST than FT concentrations and similar or even lower SA4 concentrations in both groups. Both FA4:FT and SA4:ST ratios were lower in hirsute women, except for the FA4:FT ratio in control subjects in the luteal phase. Our data are compatible with 17-hydroxysteroid oxidoreductase activity in the salivary gland. If the difference in the ratios of A4 to T between hirsute women and control subjects is attributed to this hypothetical enzymatic activity, it would suggest a more rapid conversion of A4 to T in the hirsute group.


Circulating angiotensin II levels under repeated administration of lisinopril in normal subjects. 1. To examine the effect of chronic administration of angiotensin I-converting enzyme (ACE) inhibitor on circulating angiotensin II (AII) concentration, 20 mg of lisinopril was administered once daily for 7 consecutive days to eight healthy volunteers. 2. Plasma ACE activity was inhibited to less than approximately 30% of the pretreatment level during the repeated administration. 3. Mean arterial pressure (MAP) was slightly but significantly reduced during the administration period. Plasma AII concentration measured by an established method using high performance liquid chromatography combined with a radioimmunoassay, however, was maintained at approximately the pretreatment level when it was measured at 24 h intervals after each administration of lisinopril. 4. With the gradual recovery of ACE activity following discontinuation of administration, the plasma AII concentration correlated with AI concentration (r = 0.46), and also with the product of AI and ACE activity (AI x ACE; r = 0.80), corresponding to the formula obtained from the kinetics of ACE activity. No correlation was observed between MAP and AII levels throughout the study period. 5. We conclude that in normal subjects repeatedly administered with ACE inhibitor, the AII level in the circulation is still determined by an elevated level of AI and any remaining ACE activity, thus maintaining AII at pretreatment levels. We confirmed that it is not necessary to achieve a decrease in plasma AII concentration through the chronic administration of ACE inhibitor in order to effectively lower blood pressure.


Interaction of the non-steroidal anti-inflammatory drug flufenamic acid with gastric acid secretion and H+/K(+)-ATPase. 1. The effects of the non-steroidal anti-inflammatory drug (NSAID) flufenamic acid on H+ production in isolated and enriched guinea-pig parietal cells and on H+/K(+)-ATPase activity in ion-tight inside-out membrane vesicles from pig gastric mucosa were studied. 2. At low concentrations (0.1 and 1.0 mumol/L), flufenamic acid increased the secretory response of parietal cells to dibutyryl cyclic AMP (dbcAMP). At higher concentrations (10 and 100 mumol/L) it progressively inhibited basal and dbcAMP-stimulated acid production. 3. Flufenamic acid (10 mumol/L) increased K+ (0.5-10.0 mmol/L) and K+ (0.5-1.0 mmol/L) plus gramicidin-stimulated ATPase activity in gastric membrane vesicles. The Km value for K+ (1.6 and 1.0 mmol/L in the absence and presence of gramicidin, respectively) was decreased to 0.8 and 0.5 mmol/L, respectively. At higher concentrations (greater than or equal to 50 mumol/L), flufenamic acid inhibited K+ plus gramicidin-stimulated ATPase activity (inhibited concentration at 50% [IC50] = 186 mumol/L) and reduced the proton concentration (IC50 = 50 mumol/L). 4. It is concluded that flufenamic acid-induced enhancement of dibutyryl cyclic AMP-stimulated H+ production in the parietal cell reflects the stimulation of H+/K(+)-ATPase. We suggest that activation of the enzyme involves increased affinity of K+ towards the K(+)-binding site of the enzyme and/or increased KCl permeability at the vesicle membrane. The inhibitory action of the drug on H+ production in parietal cells results from a detergent and/or protonophoric-like action at the apical parietal cell membrane, and from inhibition of H+/K(+)-ATPase activity.


[The evolution of non-A, non-B hepatitis: hepatitis C and hepatitis E]. The nosological evolution of non-A, non-B hepatitis is synthetically reviewed. After the initially complete silence from the epidemiological and diagnostic points of view, much research has been devoted to this pathology and has already yielded a number of new acquisitions even beyond simple HCV and HEV hepatitis. The problems connected with this pathology are a direct concern of medical practice in view of its frequency in the population and of its prevention where much progress has already been made.


Ocular irritative response to YAG laser capsulotomy in rabbits: release of calcitonin gene-related peptide and effects of methysergide. The Neodymium (Nd):YAG laser is commonly used in ophthalmology mainly for the posterior capsulotomy in patients with secondary cataract after extracapsular cataract extraction. A frequent side-effect following different kinds of YAG laser treatments is an acute increase in the intraocular pressure (IOP). The present study addresses the role of calcitonin gene-related peptide (CGRP) in the ocular irritative response following YAG laser anterior capsulotomy in rabbits. The YAG laser anterior capsulotomy caused an irritative response in the eye, which consisted of an increase in the IOP, miosis and breakdown of the blood-aqueous barrier. Following YAG laser capsulotomy, CGRP-immunoreactivity was found in the aqueous humour in different molecular weight forms as revealed by gel-permeation chromatography. One of the peaks coeluted with synthetic human CGRP. Methysergide attenuated the increase in the IOP and disruption of the blood-aqueous barrier, but not the miosis, following YAG laser anterior capsulotomy. The present study demonstrates the release of CGRP into the aqueous humour following YAG laser capsulotomy, and suggests that CGRP is partly causing the increase in IOP and disruption of the blood-aqueous barrier in this irritative response.


Frequency of dual infections of corneas with HIV-1 and HHV-6. In the current study, 35 pairs of corneas from asymptomatic carriers of HIV-1 and ten pairs from AIDS patients were analyzed for the presence of HIV-1 and HHV-6. The tissues were evaluated for viral antigens, transcripts, DNA sequences and intact and infectious virus. Three corneas from two asymptomatic carriers of HIV-1 and three corneas from two AIDS patients were culture positive for HIV-1. One of the three HIV-1 positive corneas from an asymptomatic HIV-1 carrier also was culture positive for HHV-6. Two of the tissue culture positive corneas from asymptomatic HIV-1 carriers and two from AIDS patients also tested positive for HIV-1 transcriptional activity by in situ hybridization. The label denoting the transcriptional activity was limited to stromal keratocytes. Most significantly, we were able to demonstrate the presence of HIV-1 particle(s) in sections and cultured PBMC from one of the HIV-1 culture positive corneas. PBMC from the same cornea also contained herpes virus particles. This report strengthens our earlier findings that HIV-1 and HHV-6 can invade corneal tissue, which emphasizes the importance of vigorous screening of corneal donors, specifically donors with HIV-1 exposure.


Extramammary Paget's disease: diagnosis and disease pattern. Extramammary Paget's disease is a rare cutaneous disorder. We report the clinical and histochemical features of five such cases. In all the cases studied, the lesions were located on the perineum of elderly women. None of the cases showed malignant proliferation invading the dermis. Recent observations suggest that extramammary Paget's disease is a disease of the apocrine glands. Immunoreactants to identify apocrine glands have been developed and can be used to confirm the diagnosis of extramammary Paget's disease.


Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.


DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties. The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.


Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands of Rhipicephalus appendiculatus, Amblyomma variegatum, and Boophilus microplus ticks. Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor(s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guinea-pigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species, Rhipicephalus appendiculatus and Amblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected female Boophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.


Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex. We investigated the effect of zonisamide, a new antiepileptic drug, on voltage-dependent Ca2+ currents in cultured neurons of rat cerebral cortex. Whole-cell voltage-clamp recordings demonstrated at least two distinct voltage-dependent Ca2+ currents: (1) a low-threshold, rapidly inactivating component, T-type Ca2+ current, which is sensitive to 100 microM Ni2+, and (2) a high-threshold, slowly inactivating (long-lasting) component, L-type Ca2+ current. Zonisamide, a new anticonvulsant effective against maximal electroshock (MES) seizures in mice reduced T-type Ca2+ current in a dose-dependent manner. The mean percentage of reduction was 59.5 +/- 7.2% at 500 microM, but zonisamide had no effect on L-type Ca2+ current. A methylated analog of zonisamide, which is ineffective against MES seizures in mice, was tested at a concentration of 500 microM, and reduced neither T-type nor L-type Ca2+ current. These findings suggest that the effects of zonisamide against MES seizures might occur through the reduction of T-type Ca2+ current. Because drugs that are effective against MES seizures are thought to prevent seizure discharge spread, T-type Ca2+ channels could underlie a cellular mechanism of spreading activity in epileptic seizures.


Relaxant effect of pituitary adenylate cyclase-activating polypeptide on guinea-pig tracheal smooth muscle. We investigated the relaxant effect of the pituitary adenylate cyclase-activating polypeptide with 27 residues (PACAP27) and with 38 residues (PACAP38) on guinea-pig tracheal smooth muscle. Both forms of PACAP showed dose-dependent relaxant effects. The EC50 of PACAP27 was 8.7 +/- 1.9 x 10(-8) M and that of PACAP38 was 6.8 +/- 1.0 x 10(-8) M. Both increased cyclic AMP levels dose dependently and the elevation of cyclic AMP preceded the relaxation of tracheal smooth muscle. There was a marked difference in the duration of action of the two peptides. PACAP38 showed a longer-lasting relaxation compared to PACAP27. Furthermore PACAP38 maintained significantly higher levels of cyclic AMP, with cyclic AMP levels at 60 min after a 5-min exposure to PACAPs (10(-6) M) being 14.0 +/- 1.4 pM/mg protein for PACAP27 and 35.9 +/- 2.4 pM/mg protein for PACAP38. These results suggest that PACAP27 and PACAP38 may be novel potent relaxants in tracheal smooth muscle and their relaxant effect might be mediated by cyclic AMP. However PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27.


Venous and arterial endothelial cells respond differently to thrombin and its endogenous receptor agonist. The effects of thrombin and a peptide mimicking the amino terminus of its receptor, Res (42-55), on vascular reactivity were compared in isolated canine blood vessels. In saphenous veins contracted with endothelin-1, both thrombin and Res (42-55) caused relaxation in rings with endothelium and contraction in rings without endothelium. In coronary arteries, thrombin caused similar responses while Res (42-55) only caused contraction. These data suggest that different thrombin receptors are present on venous and arterial endothelial cells.


Suppression of nicotinic synaptic transmission by adenosine in myenteric ganglia of the guinea-pig gastric antrum. Conventional intracellular recording techniques were used to investigate actions of adenosine on nicotinic cholinergic transmission in myenteric neurons of the gastric antrum. Adenosine or the more potent derivatives, 5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-cyclopropylcarboxamidoadenosine, 1-deaza-2-chloro-N6-cyclopentyladenosine or N6-cyclopentyladenosine reversibly and dose dependently inhibited the fast excitatory postsynaptic potentials (fast EPSPs) in 60% of the gastric neurons. Neither adenosine nor NECA affected excitatory responses to the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodine. The EC50 concentration for inhibition of the fast excitatory postsynaptic potential (EPSP) by adenosine was 55 microM NECA was a more potent inhibitor than adenosine. The specific adenosine receptor antagonists 1,3-dipropyl-8-p-sulfophenyl xanthine or 1,3-dipropyl-8-(cyclopentyl) xanthine blocked the inhibitory effects of adenosine or NECA. Fast EPSPs were enhanced by superfusion of the antagonists alone, suggestive of ongoing inhibition of nicotinic transmission by endogenous adenosine. The antagonists had no effect on resting membrane properties, excitability or antidromic action potentials. In neurons with suppression of fast EPSPs, adenosine did not suppress all cholinergic inputs to the same neuron. The results suggest that adenosine inhibits nicotinic transmission by interacting with presynaptic P1 adenosine receptors located at cholinergic release sites.


In vitro and in vivo interactions of nitrovasodilators and zaprinast, a cGMP-selective phosphodiesterase inhibitor. We have examined the interaction of zaprinast, a selective inhibitor of cGMP phosphodiesterase, with guanylate cyclase activators on vascular smooth muscle relaxation in vitro and in vivo. Isolated porcine coronary arterial rings precontracted with prostaglandin F2 alpha (PGF2 alpha) were relaxed dose dependently by the guanylate cyclase activators nitroglycerin and nitroprusside, the cGMP phosphodiesterase inhibitor zaprinast and the endothelium-dependent agent bradykinin. A 1 h pretreatment with 0.5 mM nitroglycerin shifted the dose-response curve to nitroglycerin to the right by a factor of 90, reflecting the development of tolerance. The dose-response curve to sodium nitroprusside was also affected, albeit to a much lesser degree (9-fold increase in IC50). Both zaprinast and bradykinin remained unaffected by nitroglycerin pretreatment. A 30 min pretreatment of rings with zaprinast (1 microM) had no effect on nitroglycerin- or nitroprusside-induced relaxation in control rings, but enhanced vasorelaxation to both nitrovasodilators 7- and 2-fold, respectively, in tolerant rings. Similarly, a 30 min pretreatment of rings with 0.1 microM nitroprusside enhanced zaprinast-induced vasorelaxation 4- and 8-fold, respectively, in control and tolerant rings. Similar observations were made in vivo in anesthetized spontaneously hypertensive rats where zaprinast (0.1-3.0 mg/kg i.v.), caused dose-dependent reductions in mean arterial pressure. This effect was enhanced when rats had been pretreated with nitroprusside (1 micrograms/kg per min). In comparison, in zaprinast-pretreated rats the magnitude of depressor responses to nitroprusside (0.5-5.0 micrograms/kg) was not altered, but the duration of hypotensive response to the highest dose of nitroprusside was enhanced by zaprinast. These data demonstrate an enhanced vasodilatory response of nitrocompounds in combination with peak I-selective phosphodiesterase inhibitors.


5-HT4 receptor activation induces relaxation and associated cAMP generation in rat esophagus. Changes in mechanical events and intracellular levels of cAMP induced by the activation of the 5-HT4 receptor were investigated in the rat esophagus tunica muscularis mucosae preparation. Serotonin (5-HT) and 5 methoxytryptamine (5-MOT; 5-HT4 agonist) caused concentration-related relaxation responses, while 5-carboxamidotryptamine (5-CT; 5-HT1 agonist), 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI; 5-HT2 agonist) and 2-methyl-serotonin (2-methyl-5-HT; 5-HT3 agonist) were less active. The prokinetic agents, cisapride and renzapride also induced concentration-dependent relaxation of rat esophagus which was intermediate to 5-HT and 5-MOT in potency. The relaxation was not due to activity at receptors other than the 5-HT4 since methysergide (5-HT1 and 5-HT2 antagonist) and granisetron (5-HT3 antagonist) did not block the relaxant response to 5-HT while ICS 205930 (5-HT4 antagonist) antagonized this response (pA2 = 6.45). Serotonin also caused concentration-related increases in tunica muscularis mucosae cAMP with the rank order of efficacy of 5-HT agonists in raising tissue cAMP levels reflecting their relaxant activities (5HT greater than or equal to 5-MOT greater than 5-CT greater than DOI = 2-methyl-5-HT = control). Enhancement of cAMP concentrations was also observed following renzapride treatment. This cAMP relaxation response was specific for 5-HT4 receptor activation as demonstrated by the lack of ICS 205930 inhibition of rat esophagus relaxation caused by isoproterenol, 16,16-dimethyl-prostaglandin E2 and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)


Suramin reverses non-depolarizing neuromuscular blockade in rat diaphragm. Unexpectedly, it was observed that the P2-purinoceptor antagonist, suramin (10 microM to 1 mM), reversed the muscle paralysis caused by structurally unrelated non-depolarizing relaxants. Suramin competitively reversed the blocking action of pancuronium. Both the pre- and postsynaptic blockade of nicotinic receptors by pancuronium was counteracted, as shown by the action of suramin, using train-of-four stimulation. Suramin did not affect the paralysis caused by the depolarizing relaxant, succinylcholine. The reversal action of suramin was not due to an increase in the acetylcholine concentration in the synaptic cleft, since neither the contraction of preparations partially paralysed by diminished acetylcholine release in the presence of low Ca2+ or high Mg2+ nor acetylcholinesterase activity were affected. Suramin did not affect the reduction in twitch tension caused by adenosine and potentiated the ATP-induced reduction in twitch, indicating that ATP-sensitive receptors are not involved in the reversal action of suramin. Consequently, these results suggest that the action of suramin is due to binding with a site on the acetylcholine receptor also occupied by non-depolarizing relaxants, but different from the site occupied by succinylcholine.


Spinal antinociception by Tyr-D-Ser(otbu)-Gly-Phe-Leu-Thr, a selective delta-opioid receptor agonist. The spinal antinociceptive potency of the delta-opioid receptor agonist, Tyr-D-Ser(otbu)-Gly-Phe-Leu-Thr (DSTBULET), was studied in rats. The tail flick test was used as nociceptive stimulus and the rotarod test was used to detect any motor or sedative effects. A dose-response curve was also made for the mu-opioid receptor agonist, morphine. The ED50 for DSTBULET was 0.3 micrograms (0.4 nmol) and a near 100% maximum effect was achieved with 5 micrograms (7.5 nmol). No motor or sedative effects were detected. Antinociception by DSTBULET was antagonized by s.c. naltrindole (1 mg/kg), a selective delta-opioid receptor antagonist, and naloxone (1 mg/kg), a non-selective opioid receptor antagonist. The ED50 for morphine was 0.5 micrograms (1.0 nmol) and the antinociceptive effects were not antagonized by naltrindole (1 mg/kg). The results evidence further the important role of the delta-opioid receptor in spinal nociceptive processing.


Identification of actin kinase activity in purified fragmin-actin complex. Actin kinase phosphorylates actin of fragmin-actin complex, resulting in the inactivation of the nucleation and capping activities of the complex. Fragmin-actin complex was prepared by a new purification procedure. Incubation with ATP caused inactivation of the purified complex and phosphorylation of actin of fragmin-actin complex. The detailed analysis of the complex by SDS-gel electrophoresis showed that actin kinase was co-purified with the fragmin-actin complex. Formation of such an association between actin kinase and substrate suggests that the kinase is localized on the fragmin-actin complex to efficiently regulate actin cytoskeletons.


Involvement of MAP kinase activators in angiotensin II-induced activation of MAP kinases in cultured vascular smooth muscle cells. In cultured vascular smooth muscle cells (VSMC) angiotensin II (ang II) induces tyrosine and serine/threonine phosphorylation and activation of two mitogen-activated protein (MAP) kinases. When extracts of ang II-stimulated VSMC were fractionated by Mono Q anion-exchange column chromatography, three peaks of the activities which in vitro activate inactive MAP kinases were detected. These MAP kinase activator activities were not detected in extracts of unstimulated VSMC. In vitro activation of MAP kinases by the MAP kinase activators was accompanied by tyrosine and serine/threonine phosphorylation of MAP kinases. These results suggest that the MAP kinase activators are involved in the ang II-induced phosphorylation and activation of MAP kinases in VSMC.


Molecular modelling of the structures of endothelin antagonists. Identification of a possible structural determinant for ET-A receptor binding. Computer-aided molecular modelling of the endothelin (ET-A) receptor antagonists, BQ-123 and BE-18257B, shows that they have very similar 3D structures. Parts of their 3D structures are also shown to match closely with that reported for residues 6-8 in endothelin-1. On the basis of these similarities (and with supporting evidence from literature data on endothelin structure-activity relationships) a structural determinant is proposed for ET-A receptor binding, and novel designs of peptide are suggested for providing more potent and selective ET-A receptor antagonists.


Ultrastructural and morphometric analysis of cytoplasmic structures in preimplantation pig embryos. Preimplantation pig embryos (1-, 2-, 3/4-, 16-cell stage, blastocysts) were analysed morphologically and/or morphometrically by means of electron microscopy and computer-assisted image analysis. Qualitative and quantitative changes in localization, organization, number, and area of cytoplasmic inclusions were analysed on thin sections. The structure and number of mitochondria, number of endoplasmic reticulum profiles were recorded in several individual developmental stages. Three types of cytoplasmic inclusions were described (vesicles with yolk globules, vesicles with homogeneous light inclusions, lipid droplets), each having a different topographic distribution. Quantitatively, a gradual increase in the number of endoplasmic reticulum profiles and mitochondria occurred between 4-cell stage blastomeres and blastocyst trophectoderm cells. The significant differences recorded in number, morphology and distribution of organelles in different embryonic developmental stages are discussed in relation to physiological phenomena known to occur during cleavage and compaction.


Microinjection of cloned proviral Rous sarcoma virus DNAs into Xenopus laevis one cell embryos. Plasmid pATV8 carrying Rous sarcoma virus (RSV) DNA with one long terminal repeat (LTR) or plasmid pAPrC carrying complete RSV proviral DNA was injected into fertilized eggs of Xenopus laevis. The fate of the exogenous DNAs was followed during embryogenesis. In both cases a new form of DNA, comigrating with endogenous DNA, began to appear shortly after the injection. This new form represented a linear dimer of the injected plasmid that had formed by ligation of linear monomers of the plasmid DNAs. All forms of the exogenous pATV8 DNA disappeared gradually during neurulation. No traces of virus-specific RNA were ever found in any of the following stages. The intensity of replication of plasmid pAPrC DNA in fertilized eggs was greater than that of pATV8 DNA and, in contrast to pATV8, a small amount of pAPrC persisted through the neurula till higher stages. In some experiments virus-specific DNA was even observed in 6-day-old embryos. Digestion with Nsi I and Sac I proved integration of the plasmid into the frog genome. At the stage of gastrula and at later stages a weak signal of viral RNA was also detected.


A stable line of turkey bone marrow cells transformed by the myelocytomatosis virus strain MC31. Ultrastructural characteristics and localization of the DNA replication sites. Attempts were made to characterize cells of the LSTC-SF2 line by scanning electron microscopy and transmission electron microscopy on the ultrastructural level. The virus-transformed cells are of oval, slightly elongated shape with an undulating surface. The cell nucleus is well outlined, poor in heterochromatin but with a strongly developed nucleolus. The cytoplasm is not rich in organelles except for an abundance of mitochondria with dense granules that are often found in them. With high-resolution autoradiography the DNA synthesis sites were identified mainly in proximity to the nuclear membrane and in the perinuclear spaces. The cells under study can be regarded as immature forms of the blood series and most likely as precursors of cells of the granulocyte or monocyte series.


Partial characterization of rat cell lines infected by a Rous sarcoma virus-33. Rous sarcoma virus-33 (RSV-33) was obtained from a sample of chicken Rous sarcoma which had been dried and stored in 1933. RSV-33, like the RSV-29, has the minimal number of passages beyond its isolation from chicken tumour No. 1. Our experiments demonstrated that the Rous sarcoma virus-33 was replication non-defective and was pathogenic for rats. Established rat tumorigenic cell lines express the viral genome. All three species of viral RNA were detected and v-src proteins and gag polyproteins were identified as well in cells of R9 and R74 lines. The virus can be rescued from cells of R9 and R74 lines, thus indicating that the cells are virogenic. The cells of a permanent tumorigenic line RT1 are infected but not transformed by RSV-33. Although they contain a complete proviral genome, they do not express detectable virus-specific RNA. The virus is not rescuable from RT1 cells under in vivo conditions. Proviral DNA analysis showed that the RSV-33 contained a full-length genome, including the env gene, in contrast to the RSV-29 which was found replication defective.


Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors. We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.


Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta. Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.


Type I corticosteroid receptor-like immunoreactivity in the rat salivary glands and distal colon: modulation by corticosteroids. A 167 amino acid fragment of the N-terminal domain of the human type I corticosteroid (mineralocorticoid) receptor was fused to the glutathione S-transferase gene using the Gex expression plasmid and the fusion protein used to raise the monospecific polyclonal antibody, MINREC4. Immunostaining experiments showed that MINREC4 specifically bound type I receptor in the distal tubule of the kidney, the ductal elements of the salivary glands and the epithelium of the distal colon in the rat. Adrenalectomy abolished staining in the parotid and colon, and reduced immunoreactivity in the submandibular gland. Administration of corticosterone or aldosterone resulted in partial restoration of immunostaining in the parotid, and a complete restoration of staining to intact levels in the submandibular gland and colon. These results suggest that adrenocorticoid binding to the type I receptor may result in tissue specific conformational changes in the binding protein and that the MINREC4 antibody may be used to study the effects.


Involvement of multiple sulfhydryl groups in melatonin signal transduction in chick brain. To gain insight into the molecular mechanism underlying melatonin binding and signal transduction in the chick brain, we have investigated the role of -SH groups, using a sulfhydryl alkylating reagent N-ethylmaleimide (NEM). At least two -SH groups are involved in the formation of the receptor-G protein complex: one is sensitive to and the other relatively insensitive to NEM. Alkylation of the sensitive group selectively abolishes high affinity binding of 2-[125I]iodomelatonin ([125I]MEL), similar to the effect induced by GTP, thus leading to a complete loss of sensitivity to nucleotides. Modification of both groups causes a marked reduction in binding capacity. Agonists with high affinity, but not other compounds with low affinity for the melatonin receptor, protect against alkylation by NEM. GTP gamma s does not significantly alter the reactivity of -SH groups towards NEM, but agonist-protected receptors remain sensitive to this nucleotide. Moreover, NEM pretreatment blocks the inhibitory effect of melatonin on forskolin-stimulated adenylate cyclase activity in chick brain. These data suggest that the -SH group modulating agonist affinity may lie within the coupling domain between the receptor and G protein but outside of the GTP binding site. In addition, sulfhydryl groups are essential for melatonin binding and signal transduction in chick brain.


Receptor-mediated interactions of advanced glycosylation end products with cellular components within diabetic tissues. AGEs are nonenzymatically glycosylated adducts of proteins that accumulate in vascular tissues with aging and at an accelerated rate in people with diabetes; AGEs are closely linked to tissue damage due to their high reactivity in protein cross-linking. A macrophage-monocyte receptor system for AGE moieties is shown to mediate the uptake of AGE-modified proteins by a process that also induces cachectin-TNF, IL-1, IGF-I, and PDGF secretion. Thus, in addition to removing senescent glucose-modified proteins and cells, AGE-mediated release of growth-promoting factors may represent a mechanism by which macrophages signal mesenchymal cells the need for replacement of senescent proteins. The age of the macrophage correlates inversely with the binding and removal capacity of the AGE receptor, possibly preventing the clearance of cross-linked proteins and the compounding aging-related tissue damage. In addition to monocyte and macrophages, other cells express similar receptors for AGE-proteins, including endothelial cells, fibroblasts, and mesangial cells. Endothelial cell AGE-receptors mediate transcytosis of AGEs to the subendothelium, induce increased permeability, and enhance endothelium-dependent procoagulant activity. Renal mesangial AGE receptors mediate PDGF-dependent extracellular matrix protein production. Fibroblast AGE receptors may influence cellular proliferation by EGF and EGF-receptor regulation. These findings, in connection with the known abundance of AGEs in aged and diabetic tissues, indicate that AGE-ligand-receptor interactions are crucial for the development of age- and diabetes-related vascular tissue and renal pathology.


Clinical decision analysis of HIV screening. Various health organizations, and government agencies have developed differing recommendations to address the issue of who should be tested for HIV (human immunodeficiency virus). This study used decision analysis to evaluate approaches to testing for HIV infection in a variety of clinical situations. Four decision tree branches were evaluated to determine the relative importance of HIV seroprevalence, test sensitivity, test specificity, and other factors in four testing scenarios: 1) no HIV testing of anyone, 2) mandatory HIV testing for all health care workers and hospitalized patients younger than age 65, 3) testing of persons determined to be at high risk for HIV infection based on risk assessment, and 4) voluntary HIV testing and reporting. No HIV testing results in a situation in which HIV status is unknown for all patients and health providers; issues of test sensitivity and specificity are not pertinent. Mandatory testing of health care workers and hospitalized patients, and testing only of high-risk persons, is heavily dependent on assumptions regarding HIV disease prevalence and on test sensitivity and specificity. Voluntary testing is more dependent on political decisions regarding the consequence of positive tests than it is on HIV prevalence or test characteristics. Policies regarding HIV testing are heavily dependent on the prevalence of HIV seropositivity, the sensitivity and specificity of HIV blood tests, and public policy.


[Comparison of functional results of ileorectal and ileo-anal anastomoses in familial adenomatous polyposis. Conversions of ileorectal anastomoses into ileo-anal anastomoses]. The aim of this study was to compare the functional results of ileo-rectal anastomosis and ileal pouch-anal anastomosis in a group of patients with familial adenomatous polyposis who had conversion of a ileorectostomy into a ileal pouch-anal anastomosis. In 2 cases (8.3 percent), the conversion was impossible because of abdominal desmoid tumors. For the remaining 21 patients, with more than 1 year follow-up, the number of bowel movements per 24 hours was 3.8 +/- 0.2 before and 4.6 +/- 0.3 after conversion. Daytime and nighttime continence and sensation of the need to defecate were unchanged. The number of patients having nocturnal bowel movements were higher after the pouch procedure (40 vs 10.5 percent). After ileorectostomy and after conversion, 89.5 and 80 percent of the patients had good functional results respectively. Ninety percent of the patients said that results were unchanged or improved after the conversion. In familial adenomatous polyposis the functional results of ileal pouch-anal anastomosis are similar to those of ileorectostomy but the first procedure eradicates the risk of rectal cancer. A conversion to ileal pouch-anal anastomosis should to be proposed to patients with ileorectostomy and at high risk for rectal cancer.


[Recurrence of virus B hepatitis after liver transplantation]. Thirty-three HBs antigen positive patients without signs of viral replication underwent orthotopic liver transplantation and received long term passive immunoprophylaxis with anti-HBs immunoglobulins at high doses perioperatively and then at a dose of 10,000 IU every month. All patients became negative during the first 6 months following surgery. At 34 months the survival rate was 67 percent and the actuarial recurrence rate of serum HBs antigen was 7.1 percent. Reappearance of HBs antigen was associated with evidence of HBV replication and histological alterations of the graft. In our experience, long term passive immunoprophylaxis reduces HBV reinfection of the grafted liver.


[Abnormal expression of hepatitis B virus sequences integrated in human hepatocellular carcinomas]. Although epidemiologic studies have clearly demonstrated the importance of the hepatitis B virus in the genesis of hepatocellular carcinoma, the molecular basis for this tumorigenic effect is still under debate. The finding of hepatitis B virus DNA integration into human liver DNA in many cases of hepatocellular carcinoma suggested that these integrated viral sequences may be involved in liver carcinogenesis. In an attempt to clarify this point, we studied 9 tumors which developed in non cirrhotic livers. All tumors contained viral integrations (ranging from 1 to 6 different integrants) and 4 showed abnormal hepatitis B virus mRNA (2.3 to 7.5 kilobases long). The analysis of the corresponding cDNAs revealed the existence of hybrid transcripts containing both genomic and viral sequences. In 2 cases, the viral-host junctions were mapped within the cohesive-end region of the hepatitis B virus genome leading to the production of a transcript encoding a 3' truncated X protein. In another case, the cellular sequences present in the co-transcript were located in 5' with respect to the hepatitis B virus sequences. This observation strongly suggests that, in this patient, integration took place near a cellular gene. Further analysis of this integrant should help in identifying the putative gene and its application in the development of the tumor. We conclude that the study of abnormal hepatitis B virus transcripts in liver tumors provides a positive approach to study the direct role of HBV in carcinogenesis as an insertional mutagen.


M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells. The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.


Estimation of IgG subclasses of anti-HCV (C100-3) in non-A, non-B fulminant, acute and chronic hepatitis and it's significance. To determine whether identification of subclasses of anti-HCV IgG would help distinguish between acute and fulminant hepatitis, we assayed serum IgG subclasses of anti-HCV (C100-3) in non-A, non-B hepatitis. Anti-HCV IgG was restricted to two subclasses in different diagnostic conditions; anti-HCV IgG1 and anti-HCV IgG3. Anti-HCV IgG1 was the main subclass in acute hepatitis (AH), and was positive in all the cases and only one case was positive for anti-HCV IgG3. Anti-HCV IgG3 was the dominant subclass in fulminant hepatitis (FH). Out of the total 12 cases of FH, who were positive for either anti-HCV IgG or for any of it's subclasses, in 10 cases (83%), anti-HCV IgG3 had higher optical density (OD) values than anti-HCV IgG1 and in 6 cases (50%) was the only subclass of anti-HCV IgG being positive. In 5 cases with FH, anti-HCV IgG3 became positive even when anti-HCV IgG was negative. These findings from the basis of a new observation and are likely to be helpful in the diagnosis of non-A, non-B fulminant hepatitis.


Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis. Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.


Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci. Thirteen Staphylococcus aureus and S. epidermidis strains obtained from nose and hand of two employees and one patient of a medical ward as well as two S. hemolyticus strains were analysed according to their restriction fragment length patterns (RFLP) by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI and SstII. Species identification of the isolates was performed by a system which includes 20 biochemical reactions. Furthermore, the antibiotic resistance patterns of the strains were determined. While several isolates exhibited identical antibiotic susceptibilities and biochemical profiles, differences in the RFLP were obtained. In three cases, S. epidermidis strains colonizing the skin showed an identical restriction profile as isolates from the mucous membranes of the same person. We concluded that the analysis of staphylococcal strains by PFGE is an important epidemiological tool with high discrimination power.


Chromosomal genotypes (evolutionary lines) of Salmonella berta. The copy number and location of the insertion sequence IS200, a mobile DNA element, was established across a collection of Salmonella berta. All strains contained one common site, assumed to be present in the evolutionary ancestor of this serovar. With one exception, all strains, including recent outbreak isolates from the UK and sporadic isolates of world-wide distribution, were representatives of a single genotypic clone which carried three common IS200 insertion sites. This clone has acquired diverse combinations of plasmids, reflecting its actual or recent distribution and host. A single isolate, belonging to a second, minor genotypic clone was characterised by two IS200 insertion sites.


Phospholipids of Trypanosoma cruzi: increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. We have studied the effect of carbamoylcholine in Trypanosoma cruzi epimastigote forms prelabelled with [32P]-Pi. Suspensions of cells were incubated at 28 degrees C to measure changes in the levels of [32P]-labelled phospholipids after stimulation. The presence of this cholinergic agonist induced changes in the phosphoinositide metabolism; a shift in the levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) was observed, whereas the levels of the other glycerophospholipids were not changed. This study shows that carbamoylcholine either directly or indirectly influences changes in phosphoinositide metabolism.


Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas. Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression.


Human papillomavirus types and cervical squamous intraepithelial lesions that recur after cold-knife conization. The purpose of this study was to analyze the HPV types and histological margins of cervical squamous intraepithelial lesions (SILs) treated by cold-knife conization and to correlate this with recurrent disease. Of 203 cone biopsies done for SILs primarily because the entire lesion could not be visualized at colposcopy, follow-up information was available for 85 cases. Of these 85 cases, biopsy-proven recurrences were documented for 10 (12%) women. In the SILs which recurred after conization, the lesion was noted on the surgical margin in 7/10 (70%) cases. In contrast, SILs that did not recur after cold-knife conization were detected on the surgical margin in only 12% of cases. In 7 of the recurrences, the HPV type detected in the pretreatment SIL was the same as that detected in the SIL that occurred after conization. In the other 3 recurrences, one of either the primary or recurrent SILs was HPV positive and the other corresponding lesion HPV negative. It is concluded that detection of a SIL on the surgical margin is a marker for recurrent disease and that recurrences are often associated with the same HPV type as that noted in the pretreatment SIL.


Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix. Fifty cervical adenocarcinomas and 50 squamous cell carcinomas from age-matched patients were examined for human papillomavirus (HPV) types 16 and 18. The polymerase chain reaction was used to examine formalin-fixed, paraffin-embedded carcinoma tissues for 120 and 113 bp sequences, respectively, of the highly conserved E6/E7 regions of the viral genomes. HPV type 16 was detected more often in squamous cell carcinomas than in adenocarcinomas (60% vs 18%, P less than 0.001). Conversely, HPV type 18 was detected significantly more often in adenocarcinoma tissues (52% vs 12% in squamous cell carcinomas, P less than 0.001). These differences may reflect the fact that different virus receptors exist in cervical cells with different morphologic potential, or they may indicate that the specific HPV infection actually plays a role in directing carcinogenesis.


Gonadotropins, estradiol, and growth factors regulate epithelial ovarian cancer cell growth. Indirect evidence suggests that gonadal steroids and gonadotropins may have a role in the genesis of epithelial ovarian cancer. In the studies reported herein, we established 17 beta-estradiol (E2) secreting cell cultures from an omental metastasis of an epithelial ovarian cancer. We demonstrate that human chorionic gonadotropin (hCG), human follicle-stimulating hormone, and epidermal growth factor (EGF) increased cell growth in a dose- and time-dependent manner, whereas E2 inhibited cell growth in the nanomolar range. Epidermal growth factor was able to partially block the negative effect of E2; a similar but quantitatively lesser effect was observed with hCG. These results provide direct evidence to support the view that gonadotropins, EGF, TGF beta (transforming growth factor), and estradiol may modulate growth of metastatic epithelial ovarian cancer cells.


Squamous cell carcinoma of the cervix: HPV 16 and DNA ploidy as predictors of survival. In this study, the hypothesis that DNA ploidy and the presence of HPV 16 and HPV 18 DNA affects survival of patients with squamous cell carcinoma of the cervix was tested. Archival paraffin blocks from biopsy and surgical specimens were obtained from 127 women diagnosed in 1977-1984. Determination of DNA ploidy was by flow cytometry and HPV 16 and HPV 18 DNA status by polymerase chain reaction with subsequent dot-blot hybridization. For each patient, age, stage, treatment modality, and 5-year survival were correlated with ploidy and HPV status. HPV 16 DNA was present in 53% of the tumors. HPV 18 was not detected in this population. HPV 16 DNA was found twice as often in Stages IB and IIA than in advanced-stage disease (III and IV). These advanced-stage tumors were more commonly aneuploid. Neither HPV status nor DNA ploidy were predictive of survival for any stage of disease or therapeutic modality.