Differential changes in left and right ventricular SR calcium transport in congestive heart failure. To examine the status of sarcoplasmic reticulum (SR) with respect to Ca2+ transport in congestive heart failure due to myocardial infarction, the left coronary artery in rats was ligated for 4, 8, and 16 wk. The left heart function was assessed with an intraventricular pressure transducer, and SR membrane fractions from the right ventricle and the viable left ventricle were isolated for measuring the ATP-dependent Ca2+ uptake activities. In comparison to sham-operated controls, SR Ca2+ uptake activity was decreased in viable left ventricle of the experimental animals at 4, 8, and 16 wk. On the other hand, SR Ca2+ uptake activity in the right ventricle was increased at 4 and 8 wk, but no change was apparent at 16 wk of coronary occlusion. The decrease in SR Ca2+ uptake in left ventricle and increase in right ventricle were associated with corresponding changes in maximal velocity values without any alterations in the affinity for Ca2+. These opposite changes in the right and left ventricles were dependent on the scar size as well as time after inducing the myocardial infarction. The SR Ca(2+)-stimulated adenosinetriphosphatase activity was decreased in left ventricle and increased in the right ventricle from 4 wk experimental animals. The results suggest differential remodeling of the SR membranes with respect to Ca(2+)-pump mechanisms in left and right ventricles during the development of congestive heart failure.


Comparison of renal actions of urodilatin and atrial natriuretic peptide. A 32-amino acid atrial natriuretic peptide (ANP)-like peptide, putatively synthesized by the kidney, has recently been isolated from human urine. This peptide, urodilatin (Uro), is structurally similar to the 28-amino acid ANP, suggesting that they might have similar actions on renal fluid and electrolyte excretion. The purpose of this study was to characterize the direct renal actions of low doses of Uro infusion and to compare them with the effects of equimolar intrarenal infusions of either ANP or the 24-amino acid atriopeptin III (AP III). Synthetic Uro was infused into the renal artery of pentobarbital sodium-anesthetized mongrel dogs (n = 8) at 0.14, 0.28, and 1.43 pmol.kg-1.min-1 while renal perfusion pressure was servo-controlled at 100 mmHg. Uro infusion at 1.43 pmol.kg-1.min-1 increased sodium excretion from an average control of 57.4 +/- 10.1 to 159.0 +/- 24.4 mueq/min. Uro infusion at the highest dose also increased potassium excretion (28.0 +/- 4.5 vs. 40.4 +/- 7.4 mueq/min), chloride excretion (56 +/- 11 vs. 155 +/- 22 mueq/min), and urine volume (0.54 +/- 0.12 vs. 1.22 +/- 0.25 ml/min). Fractional lithium excretion, a marker for proximal tubular sodium reabsorption, was not altered by Uro infusion, nor were urinary guanosine 3',5'-cyclic monophosphate excretion, glomerular filtration rate, or effective renal plasma flow changed. Equimolar infusions of these low doses of either alpha human ANP (n = 6) or AP III (n = 8) had no effect on any of the measured variables. Thus, within the range of doses used in this study, Uro was a more effective natriuretic and diuretic agent than either ANP or AP III.(ABSTRACT TRUNCATED AT 250 WORDS)


Preoptic norepinephrine-induced hypothermia is mediated by alpha 2-adrenoceptors. We have shown previously that norepinephrine (NE) microdialyzed into the preoptic area of conscious guinea pigs evokes a fall in core temperature (Tco) that is mediated by a reduction in metabolic rate. To identify the adrenoceptor subtype(s) involved in this effect, we microdialyzed intrapreoptically various adrenergic agonists or antagonists singly or in combinations. Tco and ear skin temperatures of the animals were monitored throughout the experiments. alpha 1-, beta-, beta 1-, and beta 2-agonists and antagonists did not induce significant Tco changes. Although the alpha 2-antagonists yohimbine (Yoh) and rauwolscine (Rau) did not have thermal effects per se, the alpha 2-agonist clonidine evoked dose-dependent Tco falls that were abolished by codialyzed Yoh and Rau. The microdialysis of NE evoked, as before, a 0.7 +/- 0.2 degrees C Tco fall; it was abolished by the codialyzed alpha-antagonist phentolamine, Yoh, and Rau but not by the beta-antagonist propranolol. No adrenoceptor agonist induced changes in ear skin temperature. These results indicate that the hypothermizing effect of intrapreoptically microdialyzed NE is achieved by a reduction in metabolic heat production, mediated by alpha 2-adrenoceptors.


Fetal iron and cytochrome c status after intrauterine hypoxemia and erythropoietin administration. Chronic fetal hypoxemia stimulates erythropoiesis and may result in a redistribution of fetal iron from plasma into erythrocytes. We studied the response of fetal plasma erythropoietin (Ep) to hypoxemia, the role of Ep in stimulating erythropoiesis in utero, and the effect of augmented erythropoiesis on fetal plasma Ep and iron and tissue cytochrome c concentrations in 19 chronically instrumented late-gestation fetal sheep. The fetuses were stimulated to produce 28 erythropoietic responses after exposure to 1) acute hypoxemia (1-5 days), 2) chronic hypoxemia (greater than 7 days), and/or 3) administration of 1,500 U recombinant human Ep concurrently during normoxemia. Plasma Ep peaked less than 12 h after the onset of hypoxemia or Ep bolus. Plasma iron decreased 24-48 h later and returned to baseline 48-96 h after normalization of Ep levels to baseline. The plasma iron response was directly related to the erythropoietin stimulus (r = 0.79, P less than 0.001) and inversely related to liver iron concentration at death (r = -0.84, P less than 0.001). Nine fetuses with depleted liver iron concentrations at autopsy had significantly lower heart and skeletal muscle iron concentrations compared with animals with 10% of control liver iron remaining. Skeletal muscle and heart iron and cytochrome c concentrations were significantly correlated. Ep has a potent biological effect on fetal erythropoiesis and iron metabolism. Augmented fetal erythropoiesis, mediated by Ep, results in decreased plasma iron, hepatic storage iron, and skeletal and cardiac muscle iron and cytochrome c. The model potentially explains the iron abnormalities found in newborn infants after fetal hypoxia.


Captopril enhances renal responsiveness to ANF in dogs with compensated high-output heart failure. The systemic hemodynamic, hormonal, and renal effects of chronic angiotensin-converting enzyme inhibition (CEI) with captopril and the responses to synthetic atrial natriuretic factor (ANF) infusions in the presence and absence of captopril were examined in normal dogs (n = 6) and in dogs with an arteriovenous (AV) fistula and compensated high-output heart failure (n = 6). This experimental model is characterized by normalization of the circulating renin-angiotensin-aldosterone system (RAAS) and persistent elevations in central filling pressures and plasma ANF. In both normal and AV-fistula dogs, oral captopril for 1 wk at 35 mg.kg-1.day-1 in three divided doses produced progressive reductions in arterial and atrial pressures (P less than 0.05), plasma ANF (P less than 0.05), and aldosterone (P less than 0.05). After 1-2 days of a modest increase in urinary sodium excretion (UNaV) (P less than 0.05), all of the dogs regained and maintained sodium balance during captopril administration. On the 8th day of the captopril regimen, synthetic ANF was infused at 15 and 30 ng.kg-1.min-1 for 75-min periods each. Control infusion experiments were performed in the same animals before captopril administration. The normal dogs exhibited dose-related elevations in UNaV (P less than 0.05) that were not augmented with captopril (P greater than 0.05). In contrast, in the AV-fistula dogs the observed renal unresponsiveness to synthetic ANF in the control experiments was reversed with chronic CEI, and ANF-induced UNaV achieved levels comparable to those obtained in the normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of Ca2+ and K+ currents by "antifreeze" proteins. For the last two decades, the research on fish "antifreeze" proteins has focused exclusively on their ability to depress noncolligatively blood plasma freezing points, presumably by binding to ice crystals. We report evidence that antifreeze polypeptides from the winter flounder (Pseudopleuronectes americanus) have another special property, the ability to block ion channels. In experiments with porcine granulosa cells we show, using the patch-clamp technique in the whole cell configuration, that these proteins suppress effectively calcium and potassium currents. The results of dose-response studies indicate a protein-protein interaction mechanism.


Rotavirus infection in children in Saudi Arabia. Three hundred sixty-three fecal specimens were collected from infants and young children with gastroenteritis over a 13-month period in Jeddah, western Saudi Arabia. Rotavirus was detected in 46% of the 363 specimens tested using an enzyme-linked immunosorbent assay (ELISA), and in 40.7% of 113 specimens using a latex agglutination test. One hundred nine of the 113 specimens that were positive by the latex agglutination test were also positive by ELISA. Electron microscopy was used to examine some specimens to demonstrate the presence of the virus. Rotavirus was detected throughout the 13-month study period, with an increase in the frequency of infection in the cooler months. Infection with this virus was more frequent among infants and children less than two-years old, with a maximum incidence among children 13-15 months old. In the 363 stool specimens tested, rotavirus was found in mixed infections with bacteria in 0.44%, with parasites in 1.31%, and with yeast in 0.66%.


Recommendations for treating leishmaniasis with sodium stibogluconate (Pentostam) and review of pertinent clinical studies. Pentavalent antimonial compounds have been the mainstay of the treatment of visceral, cutaneous, and mucosal leishmaniasis for approximately half a century. Pentostam (sodium stibogluconate) is the pentavalent antimonial compound available in the United States (through the Centers for Disease Control). As dosage regimens for treating leishmaniasis have evolved, the daily dose of antimony and the duration of therapy have been progressively increased to combat unresponsiveness to therapy. In the 1980s, the use of 20 mg/kg/day (instead of 10 mg/kg/day) of antimony was recommended, but only to a maximum daily dose of 850 mg. The authors have concluded on the basis of recent efficacy and toxicity data that this 850-mg restriction should be removed; the evidence to date, which is summarized here, suggests that a regimen of 20 mg/kg/day of pentavalent antimony, without an upper limit on the daily dose, is more efficacious and is not substantially more toxic than regimens with lower daily doses. We recommend treating all forms of leishmaniasis with a full 20 mg/kg/day of pentavalent antimony. We treat cutaneous leishmaniasis for 20 days and visceral and mucosal leishmaniasis for 28 days. Our judgment of cure is based on clinical criteria.


Direct identification of Trypanosoma cruzi natural clones in vectors and mammalian hosts by polymerase chain reaction amplification. The polymerase chain reaction was used to amplify the highly variable region of the kinetoplast minicircle of Trypanosoma cruzi directly in biological samples (feces of infected Triatomine bugs, blood samples of experimentally infected mice, and artificially infected human blood samples). Hybridization of the amplified DNAs with reference stocks representing different genotypes (natural clones) enabled us to characterize the stocks infecting the biological samples under study. The main interest of this new approach is the diagnosis of T. cruzi infection and simultaneous direct identification of the different natural clones circulating in vectors and mammalian blood without isolation of the stocks. The suitability of this technique for epidemiologic studies is also discussed.


Liver transplantation in malignant primary hepatic neoplasms. Between April 1986 and August 1990, 151 liver transplantations were performed at our institution, 16 (11%) of them in 14 patients with primary hepatic tumors. There were 12 hepatocellular carcinomas, 1 angiosarcoma, and 1 Klatskin tumor. None of the tumors was resectable, and there was no preoperative evidence of extrahepatic tumoral extension. Exploratory laparotomy was performed prior to transplantation in three patients and selective embolization of the tumor in six patients. There was no difference in the intraoperative requirements for blood or plasma in the patients with hepatic tumors when compared with other transplant recipients (28.6 +/- 23.6 units packed red blood cells [PRBC] versus 20.1 +/- 17.8 units PRBC, and 17.9 +/- 12.2 units plasma versus 17.1 +/- 10.5 units plasma, respectively). Extracorporeal venovenous bypass was used in all but one patient. There was no significant differences in the incidence of acute rejection or in the length of hospitalization in these patients when compared with other transplant recipients. All patients received triple immunosuppressive therapy (corticosteroids, azathioprine, and cyclosporin A). Intraoperative mortality was zero. At a mean of 13.3 months' follow-up (range: 1 to 47 months), 2 of 14 patients had died of sepsis and 1 of terminal cirrhosis (autopsies revealed no evidence of tumor recurrence); 3 patients (21%) had recurrences of the tumor (1 in the central nervous system and liver, and the other 2 in the lung). One of the three patients with a recurrent tumor is still alive after 16 months. The remaining nine patients (64%) are still alive.


A new technique of hepatectomy using an occlusion balloon catheter for the hepatic vein. To minimize intraoperative bleeding and allow more accurate resection, we have devised a new technique for hepatectomy. In addition to occlusion of the afferent vessels, we occlude the hepatic vein at the hepatocaval junction using a balloon catheter inserted transhepatically under intraoperative ultrasonic guidance. We have performed eight hepatectomies using this method. A sequence of 15 minutes of vascular occlusion followed by 5 minutes reperfusion was repeated throughout the operation, and the total ischemic time ranged from 45 to 90 minutes. This method greatly decreased intraoperative bleeding without causing significant elevation of the postoperative transaminase or lactic dehydrogenase levels when compared with occlusion of only the afferent vessels or nonischemic resection using a microwave tissue coagulator in patients undergoing equivalent resections. Also, the postoperative prothrombin time recovered to a significantly higher level, and there were no fatal postoperative complications using this method. Our method is useful for systematic hepatic resection along the hepatic vein or for resection of tumor sited at the confluence of the hepatic vein.


Ethanol self-administration in deprived rats: effects of Ro15-4513 alone, and in combination with flumazenil (Ro15-1788). Previous work in our laboratory demonstrated that Ro15-4513, a partial inverse benzodiazepine agonist, decreases self-administration of ethanol (ETOH) in rats maintained on a two-bottle regmine of a saccharin ethanol solution (ES) and water over a 35-day consumption period. The present study extended the consumption period to 60 days and examined the effects of Ro15-4513 (2.5 mg/kg), flumazenil (Ro15-1788) (8.0 mg/kg), and Ro15-4513 in combination with Ro15-1788 on the time course of ETOH self-administration. High initial intake of ES observed during the first 4 weeks declined significantly over subsequent weeks. Ro15-4513 pretreatment, however, resulted in significant reduction of ES, while significantly preventing the "normal" reduction of consumption as was observed under control conditions. The antagonistic actions of Ro15-4513 were blocked/attenuated by the benzodiazepine receptor antagonist, Ro15-1788, independent of whether consumption of the ES was low or high. Both Ro15-4513 and Ro15-1788 affected water intake differentially compared with vehicle-injected controls. The results suggest that GABA-benzodiazepine mechanisms may be important in altering chronic ETOH drinking patterns depending upon experience with ETOH, tolerance, or learning.


Outcomes of co-morbid alcoholic men: a 1-year follow-up. In this prospective, 1-year study, 360 males admitted to an inpatient alcoholism treatment program were administered a DSM-III compatible structured interview and subtyped by co-occurring psychiatric disorder. Forty percent satisfied diagnostic criteria for alcohol dependence while 27% met criteria for alcohol dependence and one additional psychiatric syndrome. The dually diagnosed patients were divided into: alcohol dependence plus drug abuse, alcohol dependence plus antisocial personality and alcohol dependence plus depression. These subtypes were compared on multiple dimensions at intake and at 1-year follow-up. At follow-up, all groups showed significant improvement in drinking and psychosocial functioning. The results suggest that subtyping alcoholics by co-morbid psychiatric disorders may be a good postdictor of clinical history, but a poor predictor of drinking outcome.


Delayed thermoregulatory changes in the immature rat following a single injection of ethanol. Adult rats exhibit rebound hyperthermia within 24 hr following a single injection of ethanol (EtOH). Tests were conducted to determine whether similar changes in thermoregulatory ability occur in the immature rat. Animals were administered saline or EtOH (4 g/kg BW; intraperitoneally) at 2 to 3, 8 to 9, or 14 to 15 days of age. Littermates were handled or left undisturbed with the dams to serve as controls. All rats were tested at 24 or 48 hr post-treatment to measure steady-state colonic temperature (Tco), tail skin temperature and metabolic rate (MR) at both thermoneutral and cold ambient temperatures (Tas). The youngest group exhibited no delayed change in body temperature or MR at 24 or 48 hr post-treatment with EtOH. Likewise, thermoregulatory ability of rats pretreated with EtOH at 8 or 9 or 14 to 15 days of age was not significantly different from controls when tested 24 hr post-treatment at thermoneutral Ta. In contrast, Tco of EtOH-treated rats in the two older age groups was 1 degree C above control level when tested 24 hr post-treatment at cold Ta. This Tco response can be explained by differences in heat transfer to the tail and MR. No altered response to cold Ta was found at 48 hr postinjection, indicating recovery from the EtOH effect. A single injection with EtOH at 2 to 15 days of age results in a change in Tco, which is dependent on postinjection time, age, and Ta.


Ethanol decreases cytosolic-free calcium ions in vascular smooth muscle cells as assessed by digital image analysis. Effects of ethanol on intracellular-free Ca2+ concentration in cultured rat aortic smooth muscle cells were examined by digital imaging fluorescence microscopy using the Ca2+ fluorescence indicator, fura-2. Ethanol induced dose-dependent decrements in cytosolic-free Ca2+ concentration at 45 mM and 90 mM, which was consistent with previously reported observations of relaxation in intact rat aortic tissues. However, ethanol at high pharmacological concentrations (e.g., 450 mM) failed to induce any further inhibition in cytosolic-free Ca2+ concentration. Our results suggest that the vasodilator effects of ethanol, observed on intact blood vessels, may result in part from an interference with the availability of Ca2+ for excitation-contraction coupling in vascular smooth muscle cells.


Beneficial effects of methionine on myocardial hemodynamic and cellular functions in hemorrhagic shock. Hypochlorous acid (HOCl), produced by activated polymorphonuclear (PMN) leukocytes, has been reported to depress cardiac function and contractility. Various mechanisms exist for activation of PMN leukocytes during hemorrhagic shock and reperfusion. In order to determine the role of HOCl in hemorrhagic shock and reinfusion, the authors studied the effects of shock and reinfusion on the cardiac function, contractility, blood lactate, blood gases, and creatine kinase (CK) and MB fraction of CK (MBCK) with and without methionine (quencher of HOCl) in anesthetized dogs. Dogs were divided into two groups: Group I, hemorrhagic shock (two hours) and reinfusion (two hours): Group II, hemorrhagic shock and reinfusion with methionine treatment. Cardiac index, mean arterial pressure, and index of cardiac contractility were similar in the two groups during shock. Postinfusion recovery of cardiac function and contractility was better in group II. Increases in blood lactate were similar in the two groups during shock. The rate of return of blood lactate to preshock values after reinfusion was greater in group II. The increases in serum CK and MBCK of the two groups during shock were similar but not significant. Following reinfusion the levels of these enzymes increased significantly, but the increases in group II were less. These results suggest that HOCl produced by activated PMN leukocytes may play a role in cardiac damage during hemorrhagic shock and reinfusion. Methionine may have beneficial effects in the hemodynamic and metabolic recovery and may reduce cellular damage during hemorrhage shock and reinfusion.


The early detection of nonpalpable breast carcinoma with needle localization. Experience with 500 patients in a community hospital. During a 5-year interval, 500 women underwent 530 needle localizations (NL) for nonpalpable, mammographically suspicious breast lesions. These lesions were localized using the Homer Mammalok Breast Needle/Wire Localizer (Namic; Glens Falls, NY). Almost one half the NL (260/530) were performed for suspicious calcifications; 19 per cent (49/260) proved to be cancerous. Cancer (CA) was identified 90 times in 88 patients (17%); 64 per cent (58/90) were invasive and 36 per cent (32/90) were noninvasive carcinomas. Eighty per cent of the lesions were 1 cm or smaller. Eighty-four axillary dissections were done and only 7 patients (8%) had axillary metastasis. During the same time interval, 277 women underwent axillary dissection, with or without mastectomy, for palpable breast cancer. Ninety-one per cent (252/277) had invasive carcinomas and 38 per cent (104/277) had axillary metastasis. Estrogen receptor assays (ERA) and progesterone receptor assays (PRA) were similar for both patient populations. The authors conclude that NL is an effective, safe method of detecting highly curable, occult breast cancer. It can be done effectively at a community hospital with results very similar to large university studies.


[Male genital examination: the usefulness of penis endoscopy and the acetic acid test for the detection of papillomavirus lesions]. This study was based on 2,400 genital examinations performed by means of the acetic acid test looking for human papillomavirus (HPV) lesions in the male partners of women with HPV genital lesions. These peniscopies demonstrated HPV lesions in 56% of the men examined. In 109 cases, histological and virological examination revealed that 30% of them had areas of intraepithelial neoplasia of the penis, associated with potentially oncogenic papillomavirus infection. The value of this examination is to detect HPV lesions and, in particular, dysplastic lesions. The treatment of these lesions appears to allow a reduction in the incidence of recurrence of HPV lesions in women after treatment.


[Pharmacological approach to the platinum compounds]. Cis-Diamminedichloroplatinum (II) (cisplatin) is one of the key drugs in the treatment of solid tumors. Cis-Diammine-1, 1-cyclobutanedicarboxylateplatinum (II) (carboplatin) and cis-diammine (glycolato)-platinum (254-S) are second-generation platinum-coordination complexes developed in recent years not only to reduce nephrotoxicity but also to have antitumor activity equivalent or superior to cisplatin. Comparative pharmacological study among these three compounds was performed. Six different small cell lung cancer (SCLC) and six non-small cell lung cancer (NSCLC) lines were exposed to different concentration of the three platinum compounds, cisplatin, carboplatin and 254-S in colony assay. The IC50 values for cisplatin, carboplatin and 254-wS in SCLC cell lines were significantly lower than those in NSCLC cell lines. In addition, the IC50s for carboplatin were significantly higher than those for cisplatin and 254-S in both SCLC and NSCLC lines. A total of 15 patients entered the pharmacological study. In all, 80 mg/sqm cisplatin, 450 mg/sqm carboplatin, and 100 mg/sqm 254-S were each given to five patients by intravenous drip infusion over 30 min. Ultrafilterable platinum declined biexponentially for carboplatin and 254-S, whereas the free platinum of cisplatin fitted to a monoexponential equation. We reported the equation between nadir platelet count (NPC) and Ccr, by retrospective analysis in 38 "Training Set" patients; [NPC] = 2,783.4 x [Ccr.]- [NPC] = 2,783.4 x [Ccr.]- 64,264.7. To evaluate prospectively the equation in the "Test Set" patient and to refine it. Thirty four patients who entered phase II study of 254-S for NSCLC were prospectively analysed. Significant correlation was observed between observed NPC and predicted NPC which was calculated by the equation (R = 0.51). To refine the equation, all patients in both "Training Set" and "Test Set" were reanalyzed. Simple linear least model is shown as the best fit and refined equation is as follows: [NPC] = 2,201.7 x [Ccr.]-17,695.0. Bioassay was achieved by clonogenic techniques using NCI-H-69 (SCLC cell line) and PC-9 (NSCLC cell line) as target. Biological comparison was performed on the basis of the antitumor activity of patient's plasma using the antitumor index (ATI). The ATIs obtained by bioassay showed better correlation than the AUCs obtained by chemical assay with the clinical response for the three agents against SCLC and NSCLC according to the following equation: [Reported Response(%)] = 11.5668 + 0.0014 x [ATI] (r = 0.97).


[Intra-arterial chemotherapy with ACNU in the treatment of malignant gliomas]. Twelve patients with supratentorial gliomas were treated with intra-arterial ACNU followed by intravenous maintenance chemotherapy. Six of the patients, treated from February 1987 through May 1989, received doses of 120-200 mg/m2 (high dose treatment group); six patients, treated from June 1989 through October 1990, received doses of 80-100 mg/m2 (low dose treatment group). The drug was given via the supraophthalmic portion of the internal carotid artery or vertebral artery at a rate of 10 mg/min. The response rate (CR + PR) on CT scan was 50% (3/6) in the high dose treatment group and 17% (1/6) in the low dose treatment group. Survival periods from first operation, however, did not differ between the high and low dose treatment groups. Bone marrow suppression was dose-related and reversible at these doses. Severe neurotoxicities (hemiparesis and aphasia) developed in the patients who received 200 mg/m2 of ACNU; in one case, this was irreversible. High dose intra-arterial ACNU chemotherapy is effective in reducing tumor size on CT scan but the prognosis is not improved. The dose limiting factor of intra-arterial nitrosourea is neurotoxicity, and doses of more than 200 mg/m2 are considered to be dangerous.


Surgical treatment of ductal carcinoma in situ of the breast. 10- to 20-year follow-up. Between 1967 and 1977, 36 patients received treatment at the Virginia Mason Medical Center in Seattle, Wash, for ductal carcinoma in situ of the breast. Twenty-five patients had modified radical mastectomies, 10 had radical mastectomies, and one had a simple mastectomy. Twenty-seven patients have been followed up for at least 10 years and are without known recurrence (mean follow-up, 17.7 years; range, 8 to 24 years), eight patients died without known recurrence (mean follow-up, 10.6 years; range, 6 to 14 years), and one patient with a prior contralateral mastectomy for infiltrating cancer of the breast had a recurrence in the scalene nodes on the side of the infiltrating cancer and died of metastatic cancer. No patients with ductal carcinoma in situ had local recurrences in the ipsilateral breast or chest wall, and no patients developed cancers in the contralateral breast; one patient had axillary metastasis. Twenty-eight (78%) of 36 patients had multicentric ductal carcinoma in situ in their mastectomy specimens. Twenty-three (88%) of 26 patients with comedocarcinoma-type ductal carcinoma in situ had multicentric lesions. Conversely, patients with low-grade nuclear papillary ductal carcinoma in situ did not have multicentric lesions. Five (14%) of 36 patients had incidental microinvasion discovered in the mastectomy specimens; all had comedocarcinoma. In summary, our study of patients with ductal carcinoma in situ revealed that (1) mastectomy provided excellent local and systemic control; (2) cancer in the contralateral breast was infrequent; (3) axillary metastasis was rare; and (4) histologic features of tumors markedly affected the frequency of multicentricity and chance for microinvasion.


[The polymerase chain reaction (PCR) for the detection of DNA of equine herpesviruses 1 and 4]. Formalin-fixed and Paraplast-embedded tissue samples of 42 aborted equine fetuses were examined by polymerase chain reaction for the presence of equine herpesvirus DNA. The used set of primers was located in the glycoprotein 13 open reading frame and allowed the amplification of both EHV 1 und EHV 4. By cleaving pattern analysis after Hinf I digestion EHV 1 could be distinguished from EHV 4. In 9 of the cases investigated EHV 1-DNA was detected. This finding is in absolute context with the results of the virological investigations.


The effects of okadaic acid and calyculin A on thrombin induced platelet reaction. Okadaic acid and calyculin A, specific and cell permeable inhibitors of protein phosphatase 1 and 2A, inhibited aggregation, secretion and delta [Ca++]i in thrombin stimulated platelets. The inhibitory effect of calyculin A (IC50: 3.6-4.8nM) was about two hundred times more potent than that of okadaic acid (IC50: 0.8-1.3 microM), which is consistent with the difference of the reported Ki values for protein phosphatase 1. These phosphatase inhibitors and PGI2 synergistically enhanced the phosphorylation of 50kDa protein (P50), which is solely related to the inhibition of platelet reaction. These results indicate that serine/threonine protein phosphatase 1 might play a role in platelet activation.


Cytochrome C oxidase-deficient mitochondria in mitochondrial myopathy. Electron microscopic cytochemistry was used to evaluate the behavior of cytochrome c oxidase (COX) in cultured skin fibroblasts from 4 patients with decreased COX activity (Leigh encephalopathy, fatal infantile COX deficiency). In patients with Leigh encephalopathy, all mitochondria reacted to COX staining either equivocally or negatively, indicating that all mitochondria were abnormal in these patients. In 1 patient with fatal infantile COX deficiency, intercellular heterogeneity of mitochondria was observed by COX staining. In another patient with fatal infantile COX deficiency, intracellular heterogeneity of mitochondria was observed. Patients with Leigh encephalopathy appeared to have a different type of mitochondrial COX deficiency than those with fatal infantile COX deficiency. Our result suggest that these 2 diseases may result from different genetic mechanisms.


The size of small cell lung carcinoma cells. Ratio to lymphocytes and correlation with specimen size and crush artifact. The size of small cell lung carcinoma (SCLC) cells has often been ambiguously defined as one and a half to four times that of a lymphocyte. The purpose of this study was to determine the ratio of nuclear diameter (ND) of SCLC cells to that of lymphocytes in the same tissue sections and to assess whether the size of SCLC cells correlates with the size of tumor specimens and crush artifact. The overall mean ND (microns +/- SD) of SCLC cells was 9.2 +/- 2.1, found in 36 oat cell carcinomas (OAT, 1,800 nuclei) and 16 intermediate cell carcinomas (INT, 800 nuclei). The mean ND of OAT and INT cells was 8.1 +/- 1.3 and 11.6 +/- 1.5, respectively. The mean ND of lymphocytes (2,600 nuclei) was 5.2 +/- 0.3. The overall mean of ND ratios (+/- SD) between SCLC cells and lymphocytes was 1.8 +/- 0.4 (median, 1.7), 1.6 +/- 0.2 for OAT and 2.2 +/- 0.3 for INT. The mean size of the 52 SCLC biopsy specimens was 0.6 +/- 0.9 cm. Of all the biopsies, 84.6% (n = 44) showed various degrees of tissue crushing. The ND of SCLC cells was associated with specimen size (P = .004) and the degree of tissue crushing (P = .001). Therefore, our findings further support the hypothesis that OAT should be considered the effect of artifact rather than a true variant of SCLC and that the ND of SCLC cells is approximately two times that of lymphocytes.


Evoked potentials in children with oxidative metabolic defects leading to Leigh syndrome. We studied evoked potentials in 15 children (age range: 2 wks to 4 yrs; mean: 10 mos) with metabolic disturbances that led to Leigh syndrome. These disturbances included deficiencies of pyruvate dehydrogenase (N = 5), complex 1 (N = 7), complex 4 or cytochrome oxidase (N = 2), and complex 5 (N = 1) deficiencies. Subsequent studies were performed in 11 children. All of the children with pyruvate dehydrogenase deficiency had abnormal brainstem auditory evoked potentials (BAEPs) due to poor morphology and reproducibility of the waveforms; central conduction time was normal in 4 of 5 initial studies. The patients with complex 4 or cytochrome oxidase deficiency had abnormal BAEPs, due to increased interpeak latencies and low amplitude or absent waves IV/V. Six of 7 of the children with complex 1 deficiency had normal BAEPs. The remaining patient (the youngest, age 6 wks) had only waves I and II bilaterally and suffered from the rapidly progressive form of complex 1 deficiency; the other 6 with complex 1 deficiency had the slowly progressive form. The one patient with complex 5 deficiency had normal BAEPs when first tested at 4 mos; abnormal BAEPs with loss of later waves were observed 10 weeks later. The visual evoked potentials and somatosensory evoked potentials usually were abnormal in these patients, but the findings were not specific to the patient subgroups. In all but one patient, subsequent studies disclosed a lack of normal maturational changes and/or deterioration across all 3 modalities. The BAEPs appeared to covary with the specific metabolic findings in these patients and with the patient's clinical course, but no BAEP could be considered characteristic of Leigh syndrome.


Pre-eruptive varicella encephalitis and cerebellar ataxia. Varicella-related neurologic symptoms usually appear during or following the exanthem. Pre-eruptive neurologic manifestations are extremely rare. We report a 6-year-old boy who developed encephalitis, characterized by drowsiness and left-sided hyperactive deep tendon reflexes and cerebellar ataxia, both of which antedated the exanthem by 16 days. The diagnostic and public health implications are discussed.


Cytomorphometric analysis of small cell neoplasms of the lung from specimens obtained via bronchoscopy. Primary neoplasms of the lung composed of small cells consist of undifferentiated small cell carcinoma (UCS), carcinoid tumors (CT) and some non-small cell carcinomas (NonSC) that lack cytoplasmic differentiation. The cytologic identification of tumors from specimens obtained via bronchoscopy sometimes presents difficulties due to overlapping patterns. In order to define additional differentiating criteria, we evaluated Papanicolaou-stained smears from 30 histologically proven cases, 10 from each of the three groups, with statistical analysis of computer-assisted morphologic measures. We identified significant differences between NonSC versus USC and NonSC versus CT with regard to nuclear and cytoplasmic dimensions (P less than .0001) but not between USC and CT. The nuclear:cytoplasmic ratios were similar in all three groups (P = .39). From this preliminary study we conclude that morphometric analysis can distinguish NonSC from USC and CT but not USC from CT.


Opsoclonus-myoclonus with Beckwith-Wiedemann syndrome and hepatoblastoma. The association between Beckwith-Wiedemann syndrome and hepatoblastoma is well established and relatively commonplace. The occurrence of opsoclonus-myoclonus syndrome in individuals with occult neoplasia is also well documented. However, the development of opsoclonus-myoclonus syndrome in an infant with Beckwith-Wiedemann syndrome and hepatoblastoma has not been reported previously. The list of underlying causes of opsoclonus-myoclonus syndrome should be expanded to include hepatoblastoma, particularly in any child with features suggestive of Beckwith-Wiedemann syndrome.


Calcium antagonists suppress experimental allergic neuritis (EAN). We examined the influence of Ca++ antagonist drugs on immune response and the clinical course of experimental allergic neuritis (EAN). The Ca++ antagonists verapamil and flunarizine suppressed actively induced EAN in Lewis rats in a dose-dependent fashion when given continuously by osmotic pumps from the day of immunization. If given from onset of clinical signs, day 10 after immunization, verapamil alone had therapeutic effects. The beneficial effects of the drugs seem to be mediated primarily by an action on the autoimmune response, since in vitro lymphocyte proliferation in response to nervous tissue antigens was dose-dependently inhibited by both drugs, whereas the in vitro conductivity of demyelinated sciatic nerve was not significantly affected by the Ca++ antagonists. The suppressive effect on lymphocyte proliferation could partially be bypassed by protein kinase C activating phorbol ester alone or in combination with the Ca++ ionophore ionomycin, confirming a non-toxic effect of the Ca++ antagonists on lymphocytes.


Itraconazole compared with amphotericin B plus flucytosine in AIDS patients with cryptococcal meningitis. We conducted a comparison of itraconazole versus amphotericin B plus flucytosine in the initial treatment of cryptococcal meningitis in patients with AIDS and established the efficacy of itraconazole as maintenance treatment. The trial was a prospective, randomized, and non-blinded study. The study was performed at an academic centre for AIDS, Amsterdam, The Netherlands. Twenty-eight HIV-1-seropositive men with a presumptive diagnosis of cryptococcal meningitis, randomized between 5 February 1987 and 1 January 1990, were included for analysis. Oral itraconazole (200 mg twice daily), versus amphotericin B (0.3 mg/kg daily) intravenously plus oral flucytosine (150 mg/kg daily) was administered for 6 weeks followed by maintenance therapy with oral itraconazole (200 mg daily) to all patients. Outcome measures were a complete or partial response, recrudescence and relapse. A complete response was observed in five out of the 12 patients who completed 6 weeks of initial treatment with itraconazole versus all 10 patients who completed treatment with amphotericin B plus flucytosine (P = 0.009). A partial response was observed in seven out of the 14 patients assigned to itraconazole. During maintenance therapy, recrudescence (n = 6) or relapse (n = 1) occurred in seven out of the 12 patients initially assigned to itraconazole, whereas two relapses occurred among nine patients initially treated with amphotericin B plus flucytosine (P = 0.22); recurrence of clinical symptoms was significantly related to a positive cerebrospinal fluid culture at 6 weeks (P = 0.003). Itraconazole is less effective compared with amphotericin B plus flucytosine in achieving a complete response in initial therapy in AIDS patients with cryptococcal meningitis.


Dexamethasone suppression of the calcitonin induced beta-endorphin, ACTH and cortisol secretion. Our previous observations have shown that calcitonin (CT) stimulates beta-endorphin, ACTH, and cortisol secretion. In order to give further information on the supposed hypothalamic pituitary involvement in this effect, we studied the influence of dexamethasone on this stimulative influence of CT. Six healthy women aged 50-65 years were investigated. All the subjects received 100 U CT salmon (Sandoz) i.v. at 0800 (0 time). Plasma beta-endorphin, ACTH, and cortisol were estimated every 30 min from -30 to 120 min by specific radioimmunoassays. The same subjects were evaluated a second time, at the same intervals, when 1 mg dexamethasone was administered per os at 11 PM the previous night and CT i.v. at 0800 the next morning. Beta-endorphin, ACTH, and cortisol levels (mean +/- SEM) rose significantly after 100 U CT from 5.6 +/- 0.17 to 16.75 +/- 1.8 pmol/L (p less than 0.001); from 39.6 +/- 6 to 88.0 +/- 3.1 pg/ml (p less than 0.0001) (from 8.7 +/- 1.3 to 19.4 +/- 0.7 pmol/L); and from 13.1 +/- 1.6 to 23.8 +/- 3.0 micrograms/dl (p less than 0.0001) [374 +/- 45 to 680 +/- 85 nmol/L], respectively. Dexamethasone suppressed almost completely the stimulatory effect of CT beta-endorphin rose from 4.9 +/- 0.12 to 6.3 +/- 1.3 pmol/L (n.s.), ACTH from 38.6 +/- 5.1 to 42.6 +/- 6.2 pg/ml (n.s.) (from 8.5 +/- 1.1 to 9.4 +/- 0.9 pmol/L) and cortisol from 0.88 +/- 0.23 to 0.88 +/- 0.18 microgram/dl (n.s.) (from 25.1 +/- 6.5 to 25.0 +/- 5.1 nmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)


Benzodiazepine receptor recruitment after acute stress in synaptosomal membranes from forebrain of young chicks: action of Triton X-100. In young chicks submitted to acute stress by forced swimming there was a significant increase in the number of the measurable [3H]-flunitrazepam receptors in synaptosomal membranes from forebrain. In addition, low subsolubilizing concentrations of Triton X-100 caused a significant increase in the measurable [3H]-flunitrazepam receptor number in synaptosomal membranes from non-stressed chicks. However, this Triton X-100 stimulatory effect was not observed when tested in synaptosomal membranes from stressed chicks. In all cases the affinity remained unchanged. This result suggest that: (i) acute stress and Triton X-100 induce receptor recruitment by enhancing [3H]-flunitrazepam accessibility to a pool of receptors which is unmeasurable either before stress or in absence of detergent; (ii) neither recruitment types are additive and they involve receptors coming from the same nonmeasurable pool; (iii) stress induces a maximal recruitment of existing benzodiazepine receptors; (iiii) the pool of nonmeasurable receptors represents about a quarter of the total in control chicks. The recruitment at a short time of stress could be interpreted in terms involving internalization; recycling or modulation of receptors but not its biosynthesis or degradation.


Cytokinesis.

Cytokinesis. The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.


The role of nutrition in cancer prevention and control. Evidence has accumulated from the study of populations and from animal feeding studies that strongly implicates dietary patterns and some specific nutrients in the process of promotion of cancers of the breast, colon, uterus and prostate. Nutritional factors may also complement smoking in the promotion of head and neck and lung cancer. Population studies indicate that lifestyle, which includes diet, tobacco use and alcohol consumption, may account for up to 70% of avoidable cancer mortality in this country. The development of effective dietary interventions to reduce some of these risks promises to reduce the age-adjusted incidence and mortality from these common forms of cancer. The biological mechanisms and hypotheses related to the principal dietary components of prevention and control of common cancers is presented together with a review of recent trials testing these ideas.


Effects of cyclosporin A and a non-immunosuppressive analogue, O-acetyl cyclosporin A, upon the growth of parent and multidrug resistant human lung cancer cells in vitro. We have studied the ability of cyclosporin A (CsA) and a non-immunosuppressive analogue, O-acetyl cyclosporin A (OACsA, B3-243) to inhibit the growth of human lung cancer cells in vitro. Using continuous drug exposure and the MTT colorimetric assay to determine cell growth we found that CsA produced partial growth inhibition at doses ranging from 0.5 to 3.0 micrograms ml-1 (0.4-2.4 microM). At progressively higher doses, complete growth inhibition and in situ cell lysis were seen. The P-glycoprotein expressing multidrug resistant (MDR) variant H69/LX4 of the small cell line H69/P was less sensitive to cyclosporins than the parent line, but this was not true of the non-P-glycoprotein expressing MDR variants of large cell line COR-L23 or adenocarcinoma line MOR. Sensitivity to OACsA was approximately 2-fold higher than that to CsA in most of the lines although not in the most sensitive line, COR-L88. Even in COR-L88, exposed to CsA or OACsA for 24 h, clonogenic cell survival was reduced only to 50%. There was no reduction in polyamine content of COR-L23 or COR-L88 cells following 48 h of exposure to CsA or OACsA. The effects on cell growth could not be inhibited by the addition of exogenous putrescine, nor could they be enhanced by the addition of alpha-difluoromethylorthinine. It does not appear therefore that inhibition of polyamine synthesis is the basis of the observed growth inhibition.


A phase II study of high dose epirubicin in unresectable non small cell lung cancer. Epirubicin (EPI), a doxorubicin analogue, is reported to have equal antitumour activity with lower cardiac and systemic toxicity. Recently, the maximum tolerated dose of this drug has been revised upwards with reported increased response rates in several malignancies. We initiated a phase II study of high-dose EPI as initial treatment for patients with advanced non-small cell lung cancer (NSCLC) (stage III and IV). Between May 1988 and November 1989, 25 patients were entered. The starting dose of EPI was 135 mg m-2, with dose attenuations and escalations of 15 mg m-2 based on mid-cycle evaluation of toxicity. Treatment was repeated every 3 weeks. Nine partial responses (36%, 95% CI: 18-57.5%) and 11 patients with disease stabilisation (44%) were observed. Median (range) time to progression was 19 (3-70) weeks. Median (range) survival is 32 (9-116+) weeks. There were no treatment related deaths. Major side effects were leukocytopenia WHO grade III/IV (23% of courses) and mucositis WHO grade II/III (15% of courses). In two patients left ventricular ejection fraction decreased greater than 15% compared to baseline values after a cumulative Epirubicin dose of 435 mg m-2, and therefore went off study. In none of the patients clinical signs of congestive heart failure were observed. We conclude from our data that high-dose EPI, contrary to previous negative studies using lower doses of EPI, ranks amongst the most active regimens against advanced NSCLC. Toxicity of high-dose EPI is moderate. Further evaluation of this compound in combination regimens is recommended.


Varicella vaccine in children with acute lymphoblastic leukemia and non-Hodgkin lymphoma. To determine the safety and efficacy of varicella vaccine, 17 children with acute lymphoblastic leukemia (ALL) and 2 children with non-Hodgkin lymphoma (NHL) receiving chemotherapy in remission were immunized with live attenuated varicella vaccine (Oka strain). Rash occurred in 7 children 7-53 days (median 24 days) after vaccination. There were 10-80 (median 20) erythematous vesicles and low-grade fever. All children required no specific treatment. There was no spread of varicella to their susceptible siblings. The control group comprised 92 ALL and 25 NHL patients receiving chemotherapy in remission who were nonvaccinated and susceptible to varicella. Over a risk duration of 80 person-years, one child of the vaccinated group developed varicella of mild degree, whereas in a risk duration of 120 person-years, 14 of the control group developed varicella. One patient died, though prompt antiviral therapy, especially acyclovir, was given to all of them. Herpes zoster of mild degree was observed in one child of the vaccinated group 65 days after vaccination. Two children of the control group developed disseminated herpes zoster. With acyclovir therapy, there was no mortality. The incidence of varicella in the vaccinated group was less than that of the control group. The difference is statistically significant (p = .0222), and the side effects of the vaccine were acceptable. Thus we conclude that the vaccine can be safely used in children with ALL or NHL under chemotherapy and can effectively protect such children from varicella.


Effect of wheat bran on excretion of radioactively labeled estradiol-17 beta and estrone-glucuronide injected intravenously in male rats. Urinary and fecal estrogen excretion were studied in male rats fed a non-fiber wheat starch diet (dietary fiber less than 1%; NF group; n = 4), a low-fiber wheat flour diet (dietary fiber 2%; LF group; n = 4) or a high-fiber wheat bran diet (dietary fiber 11.6%; HF group; n = 3). Short-term effects of the experimental diet on estrogen excretion were studied after i.v. injection of 5 microCi (0.185 MBq) of [14C]estradiol-17 beta (E2) into the tail vein of the rats fed the diets for 2 days. After 3 weeks on the experimental diets, the long-term effects were studied after injection of 5 microCi of [14C]E2 and 10 microCi of [3H]estrone-3-glucuronide (E1-gluc). The diet was found to affect estrogen excretion. The short-term effect indicated that rats fed the HF diet excreted a relatively large amount of labeled compounds in the feces during the first day after injection, while rats fed the NF or the LF diets excreted about half that amount over the same period. On the other hand, urinary excretion of labeled compounds was significantly higher in the NF and LF rats. The long-term effect resulted in steeper slopes (P less than 0.05) of the fecal excretion profiles of rats fed the HF diet as compared with rats fed the NF and LF diets, indicating an accelerated fecal excretion of labeled compounds in the HF rats. The kinetic profiles of 14C and 3H radioactivity in blood plasma indicated a fast decrease (t1/2 of less than 2 min) for both [14C]E2 and [3H]E1-gluc. It was concluded that, owing to the short-term effect of wheat bran intake, during the first 24 h after i.v. administration relatively large amounts of radioactively labeled compounds are excreted in feces of rats fed the HF diet. In contrast, excretion is lower in urine of these rats. When the microflora is adapted to the experimental diet the wheat bran diet still results in an accelerated fecal excretion of labeled compounds, which might be attributed to an interruption of the enterohepatic circulation of estrogens. This might result in lowered plasma and/or tissue estrogen levels and hence a decreased exposure of estrogen-sensitive tissue to estrogens, which might decrease risk on mammary (breast) cancer development.


Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes--coincident rise of DNA-relaxing activity in nuclear extracts. High affinity receptors (VDR) for 1,25-dihydroxycholecalciferol (calcitriol) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes (PBL). HL60 cells, expressing some characteristics of promyelocytes, can be induced to monocytoid differentiation by calcitriol. Specific nuclear translocation of [3H]calcitriol/VDR was examined after exposure of whole cells to 10(-9) M/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei. Specific nuclear translocation of [3H]calcitriol/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites/nucleus after 3 h of incubation in HL60 cells, whereas a maximum of approximately 310 binding sites/nucleus was found after 3 h in PBL. Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h. Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse (pulse/chase-experiments). No difference of VDR retention in pulse and pulse/chase-experiments was seen in PBL, where VDR halflife was approximately 30 min. No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in [3H]calcitriol. Radiolabeled hormone/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone--in contrast to identical experiments with intact cells--did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound. The activity of DNA relaxing enzymes (e.g. topoisomerases I and II) in nuclear extracts was measured using a PBR 322-relaxation-assay. Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol (final ethanol concentration 0.0001% v/v) in HL60 and PBL. The enhanced activity disappeared after 2 h in PBL, whereas it was still enhanced by 4 h in HL60. No effect was seen in ethanol treated controls. We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined, most likely due to translocation of receptor proteins after hormone binding. Translocated hormone/receptor complexes compete for a limited number of specific nuclear binding sites. Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment, thus initiating DNA-unwinding, a prerequisite of transcription initiation.


The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation. The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.


The metabolism of 2,4-dibromo [6,7-3H]17 beta-oestradiol in the rat: ring-A dibromination blocks male-specific 15 alpha-hydroxylation and catechol formation. The metabolism in the rat of 2,4-dibromo-17 beta-oestradiol (2,4-DBE2), a compound of potential use for tumour imaging and assessment, has been studied. 2,4-DB[6,7-3H]E2 was synthesized by bromination of [6,7-3H]E2 with N-bromosuccinimide, and administered (40 micrograms/kg, i.v.) to anaesthetized male and female rats. Metabolites were rapidly and extensively excreted in bile (60 and 82% of the dose over 1 and 6 h, respectively). No unchanged compound was excreted. 2,4-DBE2 was almost entirely oxidized to 2,4-DB-oestrone; which was largely eliminated as its glucuronide but partly (approx. 30%) metabolized to 2,4-DB-16 alpha-hhydroxyoesterone and, to a minor extent, 2,4-DB-oestriol. No products of either oxidative or reductive debromination were detected. Neither of the two oxidative transformations of 2,4-DBE2 in the rat, in contrast with those of exogenous E2, was sex-selective, and 2,4-DB-oestrone underwent less extensive hydroxylation than oestrone formed from E2. In female rats, the substituents selectively redirected the principal site of hydroxylation from C-2 to C-16, whereas in males they had no significant effect on the existing 16 alpha-hydroxylation but did block the major pathway, 15 alpha-hydroxylation. Thus the sexual differentiation of E2 oxidative metabolism was abolished by direct blockage causing metabolic switching to a latent reaction in the female rat and long-range inhibition of the vicinal hydroxylation in the male rat.


Polymyxin B enhances low density lipoprotein catabolism in hepatic and extrahepatic cells. We investigated the effects of polymyxin B (PMB), an antibiotic that binds to endotoxins, on the uptake and degradation of low density lipoproteins (LDLs) in HepG2 cells, a highly differentiated human hepatoma cell line. The results showed that PMB very effectively enhanced the binding, internalization, and degradation of LDL in HepG2 cells. The PMB-mediated enhancement of LDL uptake was not dependent on the LDL receptor-mediated pathway, as blockage of the LDL receptor by use of a monoclonal anti-LDL receptor antibody had no effect on the PMB-mediated cellular processing of LDL and PMB-mediated enhancement of LDL uptake did not cause an increase in cholesterol esterification. In addition, chloroquine and colchicine, which inhibit lysosomal degradation and cellular endocytosis, respectively, diminished PMB-enhanced degradation of LDL, indicating that PMB mediates uptake through a pathway similar to the LDL receptor-mediated pathway. The PMB-mediated uptake of LDL was sensitive to treatment with phospholipase C and pronase and was dependent on the presence of Ca2+. PMB caused similar changes in human skin fibroblasts, bovine smooth muscle cells, and bovine endothelial cells, which suggests that PMB-enhanced LDL uptake is a general cellular phenomenon. Our results thus indicate that PMB increases cellular catabolism of LDL through an endocytotic pathway not involving the LDL receptors.


Functional implications of cAMP and Ca2+ on prostaglandin I2 and thromboxane A2 synthesis by human endothelial cells. Human umbilical vein endothelial cells were incubated with ATP, ADP, thrombin, or ionophore A23187 for as long as 600 seconds. A statistically significant rise in the prostaglandin I2 (prostacyclin; PGI2) and thromboxane A2 (TxA2) release was observed after 45 seconds. The maximum amount of cytosolic free Ca2+ was reached between 20 and 30 seconds. A statistically significant elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels was observed only in response to ATP and ADP after 120 seconds. Furthermore, adenosine, caffeine, phorbol 12-myristate 13-acetate (PMA), and adenosine 5'-(alpha, beta-methylene)triphosphate were tested alone or in combination with ATP or thrombin. PMA inhibited ATP-stimulated eicosanoid biosynthesis but had no effect on thrombin. Of the agents used to increase cytosolic free Ca2+ concentrations, only PMA failed to provoke eicosanoid release. Similarly, only PMA failed to induce a Ca2+ flux in the absence of extracellular Ca2+. The data presented show that PGI2 and TxA2 release from human umbilical cord endothelial cells is closely associated with Ca2+ mobilized from intracellular sources. Extracellular Ca2+, as well as changes in intracellular cAMP levels, has no influence on endothelial eicosanoid synthesis, at least for short-term regulation (less than or equal to 600 seconds).


Synergistic stimulating effect between cyclic AMP and phorbol ester on plasminogen activator inhibitor type 2 production in human promyelocytic leukemia cell line PL-21. We investigated the effect of agents which raise intracellular cyclic AMP (cAMP) and protein kinase C activators on the production of plasminogen activator inhibitor type-2 (PAI-2) by cultured human promyelocytic leukemia cell line, PL-21. As previously reported, PMA, a protein kinase C activator, showed a strong stimulating effect on the PAI-2 production. 1-oleoyl-2-acetyl-sn-glycerol (OAG), another synthetic protein kinase C activator, also showed a stimulating effect, which was, however, much less than that of PMA. The agents which raise intracellular cAMP, dibutyryl cAMP, 8-bromo cAMP, prostaglandin E1, and 3-isobutyl-1-methyl-xanthine, little increased the PAI-2 production when tested alone, but showed significant synergistic effects with PMA or OAG. The synergistic effect between PMA and dibutyryl cAMP was further verified by SDS-PAGE followed by immunoblotting using a monoclonal antibody against the PAI-2. It is interesting that the up-regulation of PAI-2 by cAMP and the synergistic effect with PKC activators forms a contrast to the previous reported bi-directional regulation of endothelial PAI-1 secretion by PKC activator and cAMP.


Down regulation of alpha 2 adrenergic receptor numbers in platelets by insulin. Incubation of platelets from normal volunteers, who had not taken any medication at least for 2 weeks, with insulin (200 mu units/ml), resulted in the inhibition of the potentiation of ADP-induced platelet aggregation in the presence of (-)-epinephrine by 50-60% when compared with the control. The inhibitory effect of insulin was not related to the increased cyclic AMP level or decreased thromboxane A2 synthesis in these cells. It was found that the treatment of platelets with insulin decreased alpha 2 adrenergic receptors number of these cells from 413 +/- 92/platelet to 206 +/- 84/platelet as determined by Scatchard analysis of [3H]yohimbine binding. The affinity of the receptors (1.05 +/- 0.02 nM) remained essentially unchanged due to the treatment of platelets with the hormone (1.40 +/- 0.60 nM).


Studies on gross footpad lesions of chickens infected with avian reoviruses via the footpad route. Avian reoviruses grew well in the footpad of chickens inoculated with the viruses via the footpad route, resulting in gross footpad lesions of swelling. The gross footpad lesions induced under some different conditions were investigated for 14 days by two methods. In method A, the lesions were observed grossly and graded as lesion scores 0 to 4. In method B, they were expressed as a swelling index assessed by relative thickness of an inoculated footpad to uninoculated. Both methods are successful, and similar results were obtained. Gross footpad lesions were produced in all chickens aged 9 to 310 days, and the younger were the birds at infection, the earlier and severer were the lesions observed. The lesions began to appear late when the virus titer inoculated was low. It has been elucidated that severity of the lesions depends on virus strains, not on the sexes of chickens.


Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity. Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.


Sporadic congenital transmission of avian leukosis virus in hens discharging the virus into the oviducts. The efficacy of the albumen test for infectious avian leukosis virus (ALV) was examined in detecting congenitally transmitting hens. Seventy-three White Leghorn non-viremic hens with antibody to ALV were used. Eleven of the hens shed infectious ALV into their egg albumen, whereas only 7 of the 11 ALV-positive hens shed ALV antigens. The egg albumen test for infectious ALV was shown to be more effective in detecting the congenitally transmitting hens than that for ALV antigens. Then, twenty of the 62 hens which shed no infectious ALV into the albumen were studied for transmission of ALV to their embryos and for discharging ALV into the oviduct and vagina. Six of the 50 embryos from 4 hens were found to be infected with ALV but all of the 227 embryos from remaining 16 hens were free from the infection. Discharge of the virus into the oviduct and vagina was found both in the 4 transmitting hens and in 6 of the 16 non-transmitting hens. These results suggest that the hens discharging ALV into the oviduct, even though they do not shed ALV into egg albumen, may transmit the virus sporadically to their embryos.


[Coxsackie B4 and cytomegalovirus in patients with insulin-dependent diabetes]. The purpose of this work is to analyze the potential relationship between infection by Coxsackie and cytomegalovirus and the appearance of insulin-dependent diabetes (IDD). We have assessed complement fixing (CF) antibodies against both viruses in 22 samples of serum from other individuals, among which 11 were patients with a recent diagnosis of IDD--less than 3 months of evolution--(GP) and the remainder 11, supposed healthy individuals with similar age, sex and socioeconomic level (GC). In addition, the presence of anti-pancreatic islets antibodies (ICA) was assessed in 22 serum samples using the indirect immunofluorescence reaction. We have not observed any relationship between the presence of Coxsackie B or CMV antibodies and the appearance of IDD, since although the seroprevalence against CMV was higher in GP than in GC, such difference was not statistically significant. With respect to ICAs, they were only present in the GP samples as expected. Given the small size of the sample, this results are not conclusive and larger series need to be studied.


[Acute abdomen secondary to cytomegalovirus infection]. A case of a HIV-positive patient hospitalized with acute abdomen secondary to infection by cytomegalovirus (CMV), is presented. Infection by CMV is frequent in HIV-positive patients, with a relevant intestinal affection. However, its presentation as acute abdomen is more rare, although it has to be considered given that the demonstration of the presence of CMV and its potential pathogenic power have important therapeutic connotations. Currently, the use of diagnostic techniques based in specific monoclonal antibodies and DNA hybridization methods increases the diagnostic sensitivity of the traditional methods based on histological demonstration of the cytopathic effect and/or viral cultives.


Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.


Interleukin-5 is an autocrine growth factor for Epstein-Barr virus-transformed B lymphocytes. Because of the recent finding that interleukin-5 (IL-5) is produced by Epstein-Barr virus-transformed B lymphocytes (EBV-B cells), we performed studies to ascertain whether EBV-B cells might use IL-5 by an autocrine mechanism. EBV-B cells known to be IL-5 producers were capable of responding to addition of exogenous IL-5 by dose-related augmented proliferation. The addition of a neutralizing anti-IL-5 antibody reduced these effects and also dose-dependently inhibited proliferation and reduced viability of unsupplemented EBV-B cells, having a maximum effect at about 120 hours. In contrast, no stimulatory effect of IL-5 was noted on Burkitt's lymphoma cell lines, nor were these lines growth-inhibited by anti-IL-5 antibody. With biotinylated IL-5, (b-IL-5) second labeling with streptavidin-FITC, and flow cytometric analysis, binding of IL-5 to EBV-B cells cultured in fresh medium was demonstrated and could be competed for by excess unlabeled IL-5, suggesting the presence of IL-5-specific binding sites. Binding of IL-5 was reduced on cells cultured for longer periods before study but could be restored by extensively washing cells before labeling them with b-IL-5, suggesting that surface binding sites had become occupied by endogenously produced IL-5. These findings support a role for IL-5 in autocrine support of EBV-B cell growth.


Specific expression of the annexin VIII gene in acute promyelocytic leukemia. Since the translocation breakpoint t(15;17) (q22;q21) in acute promyelocytic leukemia (APL) occurs within the retinoic acid receptor-alpha (RARA) gene, the expression of many genes normally regulated by RARA may be affected by this translocation. To identify genes that may be aberrantly expressed in APL, a subtraction cDNA library of an APL patient with t(15;17) was constructed. A cDNA, pRD1, specifically expressed in APL was identified. DNA sequence analysis of pRD1 showed that this gene is similar to the DNA sequence of annexin VIII, a gene which encodes a vascular anticoagulant. The annexin VIII gene was assigned to chromosome 10, which indicates that specific expression of this gene in APL is not directly involved in the t(15;17) breakpoint region. We have analyzed the expression of annexin VIII gene in nine t(15;17)-positive APL patients and one APL patient with a chromosome 17q-abnormality. We found that all APL samples expressed high levels of the annexin VIII gene. Expression of the annexin VIII gene in all other leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia, was undetectable, except in one patient with acute myelogenous leukemia in which a very low level of expression was detected. Annexin VIII is highly expressed in the APL cell line, NB4. Its expression was significantly reduced after 8 hours of all-trans retinoic acid (ATRA) treatment, whereas the expression of RARA increased several-fold within 4 hours postinduction. Thus, increased expression of RARA preceded the downregulation of annexin VIII after ATRA induction, suggesting an inverse relationship between RARA and annexin VIII expression. Since increased expression of the fusion transcript was seen after ATRA induction and an APL without a t(15;17) translocation expressed high levels of annexin VIII, it appears that increased expression of annexin VIII in APL is not related to the fusion transcript. Therefore, dysregulation of the RARA gene may be related to the overexpression of annexin VIII in APL.


Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease. Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.


Itraconazole compared with griseofulvin in the treatment of tinea corporis/cruris and tinea pedis/manus: an interpretation of the clinical results of all completed double-blind studies with respect to the pharmacokinetic profile. Itraconazole is an orally active triazole antifungal which has been compared to griseofulvin in a number of double-blind trials. In dermatophytosis with a non-fixed treatment regimen for a maximum of 3 months, itraconazole 100 mg o.d. has produced a 100% mycological cure rate as compared with a 67% rate with griseofulvin 500 mg o.d. (p less than 0.01). Based on the pharmacokinetic profile, 100 mg itraconazole daily was then compared with 500 mg ultramicronized griseofulvin daily using a fixed treatment schedule of 15 days in tinea corporis and/or cruris and 30 days in tinea pedis and/or manus. In all studies in tinea corporis/cruris (n = 277), the superiority of itraconazole was shown for the clinical outcome at the last follow-up visit 2 weeks post-therapy (88 vs. 69%, p less than 0.01) and in the mycological outcome at the last follow-up visit (81 vs. 65%, p less than 0.05). In tinea pedis/manus (n = 210), the clinical response was virtually the same for the two treatment groups, but the most important finding was the mycological outcome with a significantly better result in favor of itraconazole at the end of treatment (77 vs. 61%, p less than 0.05) even more pronounced at the follow-up visit (85 vs. 48%, p less than 0.01). We conclude that itraconazole 100 mg daily in the treatment of tinea corporis/cruris and in tinea pedis/manus is significantly more effective than 500 mg griseofulvin daily when fixed treatment regimens are used. Furthermore, the best results are obtained with itraconazole 2-3 weeks after the end of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Resistance of Escherichia coli to nourseothricin (streptothricin): sensitization of resistant strains by abolition of its outer membrane resistance. The polycationic antibiotic, nourseothricin, represents a mixture of several streptothricins, mainly D and F. The molecular weight of the latter compound amounts to 486. Obviously, although very slowly, it can pass the outer membrane via the porin pores. It has been shown earlier that nourseothricin is able to generate some kind of channels into the outer membrane through which it can pass the cell wall. On the other hand, there were indications that resistant strains containing a streptothricin-inactivating acetyl transferase possess an additional protecting system, namely a reduced penetrability of the outer membrane. In this study, it could be shown that such strains indeed could be rendered sensitive by damaging the barrier function of the outer membrane.


Relation of bronchioloalveolar carcinoma to tobacco. To determine whether bronchioalveolar carcinoma is related to tobacco use. Case-control study. 11 teaching hospitals of Chicago, Long Island, New York, and Philadelphia, 1977-89. 87 patients with histologically diagnosed bronchioloalveolar carcinoma (cases) and 286 non-cancer and 297 cancer patients matched to cases on age, sex, race, hospital, and date of admission. 10% of male cases and 25% of female cases had never smoked. Relative risks of bronchioloalveolar carcinoma (as estimated by the relative odds) were greater for subjects who started smoking at a younger age, smoked for a longer time, or smoked more cigarettes per day. Relative risks decreased proportionally to the duration of smoking cessation. Smoking plays an important part in the aetiology of bronchioloalveolar carcinoma but is not the only potential cause because of the large proportion of never smokers among patients with this disease.


Low dihydropyridine receptor density in vasa deferentia of castrated rats. Radioligand binding studies in crude membrane preparations of vasa deferentia of normal rats, with the 1,4-dihydropyridine (+)-[3H]-PN200-110 (isradipine) showed typical saturation isotherms. The binding exhibited a KD of 259 +/- 60 pM and Bmax of 144 +/- 20 fmol mg-1 protein. The low KD and the stereoselective displacement of (+)-[3H]-PN200-110 binding by (+)- and (-)-PN200-110 and by nifedipine suggests that these tissues contain dihydropyridine receptors probably coupled to voltage-sensitive, L-type calcium channels. In membrane preparations from vasa deferentia from rats castrated 30 days previously the maximum specific binding was 25 +/- 10 fmol mg-1 protein, representing only 11% of total binding; thus, the calculation of reliable KD values was not feasible. These findings suggest that a testicular hormone, possibly testosterone, plays an important role in the regulation of dihydropyridine-sensitive, voltage-dependent calcium channels in the rat vas deferens.


Endothelium-dependent and -independent effects of exogenous ATP, adenosine, GTP and guanosine on vascular tone and cyclic nucleotide accumulation of rat mesenteric artery. 1. The effects of exogenous guanosine 5'-triphosphate (GTP) and guanosine on vascular tone and cyclic nucleotide accumulation of noradrenaline-precontracted endothelium-intact and endothelium-denuded rat mesenteric artery rings were compared with the effects of the known purinoceptor agonists adenosine 5'-triphosphate (ATP) and adenosine. 2. GTP (10 microM-1 mM) dose-dependently relaxed endothelium-intact mesenteric artery rings by producing a rapid initial response followed by sustained relaxation resembling the relaxant response to acetylcholine. GTP also slightly relaxed endothelium-denuded artery rings. The acetylcholine- and GTP-induced relaxations of endothelium-intact rings were attenuated by NG-nitro L-arginine methyl ester (L-NAME, 330 microM) which attenuation was reversed with L-arginine (1 mM). 3. Guanosine (10 microM-1 mM) relaxed both endothelium-intact and -denuded artery rings in a dose-dependent manner. The relaxations were more pronounced in endothelium-intact preparations and were only slightly attenuated by L-NAME (330 microM). 4. ATP (1 microM-1 mM) and adenosine (10 microM-1 mM) dose-dependently relaxed endothelium-intact and -denuded artery rings. The responses were more pronounced in endothelium-intact vascular preparations. 5. GTP (100 microM) and guanosine (100 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) accumulation in both endothelium-intact and -denuded artery rings corresponding to the relaxations observed. The concentrations of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were not affected. 6. ATP (100 microM) increased cyclic GMP concentration of endothelium-intact artery rings. The concentrations of cyclic AMP were not affected by ATP (100 microM) and adenosine (100 microM) in endothelium-intact and -denuded vascular preparations.7. These results provide evidence that exogenous GTP and guanosine relax precontracted endothelium-intact and -denuded rat mesenteric artery rings by increasing cyclic GMP accumulation. The response to GTP of endothelium-intact rings can mainly be explained by the release of endothelium-derived relaxing factor (EDRF), but that of guanosine is only partly due to EDRF, and is a combination of endothelium-dependent and -independent effects. The endothelium-independent response of GTP and guanosine is a direct, unknown effect on smooth muscle and guanylate cyclase.


Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed BK3 receptors in the guinea-pig trachea, and are blocked by HOE 140. 1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.


Myocardial hypertrophy, cardiac beta-adrenoceptors and adenylate cyclase activity during sinoaortic denervation in dogs. 1. The long-term effects of sinoaortic denervation on the development of left ventricular hypertrophy (assessed by the measurement of the ratio (R): heart weight/total body weight and LVT: left ventricular thickness), myocardial beta-adrenergic receptivity (measured by [125I]-cyanopindolol binding and adenylate cyclase activity) and plasma catecholamine levels (measured by h.p.l.c.) were investigated in three groups of dogs: normotensive controls (group 1), dogs made hypertensive by sinoaortic denervation and evaluated 1 (group 2) and 18 months (group 3) later. 2. Noradrenaline (NA) and adrenaline (A) plasma levels were 461 +/- 54 and 85 +/- 45 pg ml-1 in controls, 861 +/- 185 and 191 +/- 23 pg ml-1 in group 2 (P less than 0.05). They were normal in group 3 (426 +/- 132 and 110 +/- 16 pg ml-1). 3. R and LVT values were significantly (P less than 0.05) higher in sinoaortic denervated dogs (R = 7.7 +/- 0.1 and 7.8 +/- 0.2; LVT = 13.6 +/- 1.3 and 14.2 +/- 0.9 mm in groups 2 and 3 respectively) than in normotensive dogs (group 1: R = 6.7 +/- 0.1, LVT = 9.3 +/- 0.8 mm). 4. In group 1, the total number of beta-adrenoceptors (Bmax) was 37 +/- 11 and 29 +/- 6 fmol mg-1 protein in the left ventricle (LV) and right auricle (RA) respectively. In group 2, Bmax was significantly lower (10 +/- 3 in LV and 13 +/- 2 fmol mg-1 protein in RA, P less than 0.05) than in group 1. There was no difference between group 1 and group 3 (37 +/- 3 fmol mg-1 prot in LV and 31 +/- 3 fmol mg-1 protein in RA). 5. The percentage of beta 1-adrenoceptors was 82 +/- 4 in LV and 75 +/- 5 in RA in group 1. It was significantly lower (P less than 0.05) in groups 2 (LV: 33 +/- 6 and RA: 33 +/- 5) and 3 (LV: 59 +/- 3 and RA: 55 +/- 4). 6. Basal values of adenylate cyclase activity in LV significantly decreased after sinoaortic denervation.7. These data show that sinoaortic denervation is associated with left ventricular hypertrophy which appears early (1 month) and persists until 18 months despite the normalization of plasma catecholamine levels. The total number of myocardial beta-adrenoceptors is closely related to catecholamine levels but a selective decrease in beta 1-adrenoceptors is observed during cardiac hypertrophy. The fall in basal adenylate cyclase activity suggests that cardiac hypertrophy is associated with an impairment of transmembrane signalling.


Contributions of alpha 1-adrenoceptors, alpha 2-adrenoceptors and P2x-purinoceptors to neurotransmission in several rabbit isolated blood vessels: role of neuronal uptake and autofeedback. 1. The roles of autofeedback and neuronal uptake in neurotransmission produced by electrical field stimulation in several rabbit isolated blood vessels were examined. 2. Blocking drugs were used to separate the possible purinergic and noradrenergic contributions to the end organ response: prazosin, antagonist at postjunctional alpha 1-adrenoceptors; rauwolscine and yohimbine, antagonists at pre- and postjunctional alpha 2-adrenoceptors; alpha,beta-methylene ATP, desensitizing agent at postjunctional P2x-purinoceptors. In addition to desensitizing postjunctional P2x-purinoceptors, alpha,beta-methylene ATP potentiated the noradrenergic component of the nerve-induced responses. 3. In the presence of an intact neuronal uptake mechanism, the vessels showed different contributions of purinergic (via P2x-purinoceptors) and noradrenergic (via alpha 1-adrenoceptors and alpha 2-adrenoceptors) components to the end organ response to nerve stimulation: saphenous artery (approximately equal contributions from P2x-purinoceptors and alpha 1-adrenoceptors), ileocolic artery (mainly P2x-purinoceptors with a smaller contribution from alpha 1-adrenoceptors), plantaris vein (mainly alpha 1-adrenoceptors with a small contribution from alpha 2-adrenoceptors and P2x-purinoceptors) and saphenous vein (alpha 1-adrenoceptors). 4. The presence of alpha 2-adrenoceptor-mediated autofeedback could be demonstrated for both purinergic and noradrenergic components of the nerve-induced responses in the artery preparations. In the veins, potentiation of nerve-induced responses by alpha 2-adrenoceptor antagonists could not be studied due to blockade of postjunctional alpha 2-adrenoceptor-mediated vasoconstriction. 5. Blockade of neuronal uptake with cocaine potentiated the noradrenergic component of the nerve-induced responses. Both alpha 1-adrenoceptor- and alpha 2-adrenoceptor-mediated components were potentiated, with a relatively greater potentiation of the alpha 2-adrenoceptor-mediated component. In the case of saphenous vein an alpha 2-adrenoceptor-mediated component which was previously absent was uncovered.6. Blockade of neuronal uptake with cocaine had no effect or reduced the purinergic component of responses, the latter effect presumably due to enhanced alpha 2-adrenoceptor-mediated autofeedback.7. In the presence of cocaine, nerve-induced responses in the saphenous vein were biphasic. Rauwolscine potentiated the first phase and inhibited the second phase thus demonstrating effects of pre- and postjunctional alpha 2-adrenoceptor-mediated activation in the same preparation.8. In conclusion, neuronal uptake and autofeedback processes play important and complex interacting parts in determining the relative contributions of alpha 1,- and alpha 2-adrenoceptors and P2.-purinoceptors in the end organ response to neurotransmission in blood vessels.


The effects of dietary treatment with essential fatty acids on sciatic nerve conduction and activity of the Na+/K+ pump in streptozotocin-diabetic rats. 1. This study examined the effects of dietary essential fatty acid supplementation (5% (w/w) evening primrose oil) upon sciatic motor nerve conduction velocity and 86Rb+ pumping in sciatic nerve endoneurial preparations in rats with 4 to 5 weeks of streptozotocin-induced diabetes. 2. Control diabetic rats (dietary supplementation with 5% (w/w) hydrogenated coconut oil) exhibited a reduction in motor nerve conduction velocity (16%; P less than 0.05) compared to similarly-fed non-diabetic controls, but there was no significant alteration in ouabain-sensitive 86Rb+ pumping, a parameter reflecting activity of the Na+/K+ pump. 3. Treatment of diabetic rats with evening primrose oil prevented completely the development of the motor nerve conduction velocity deficit without affecting the severity of diabetes. Evening primrose oil treatment did not significantly affect motor nerve conduction velocity of non-diabetic animals. 4. Evening primrose oil treatment caused a significant reduction in activity of the Na+/K+ pump in sciatic nerves of diabetic animals (45%; P less than 0.05). 5. These results suggest that the acute conduction velocity defect arising in streptozotocin-diabetic rats, and the actions of evening primrose oil upon this, are independent of any effect on activity of the Na+/K+ pump. Other putative mechanisms are discussed.


Evidence for multiple endothelin receptors in the guinea-pig pulmonary artery and trachea. 1. The responses of the three peptides, endothelin 1 (ET-1), endothelin 2 (ET-2) and endothelin 3 (ET-3) were analysed on isolated circular segments of pulmonary arteries and trachea from the guinea-pig. 2. In the pulmonary artery, the vasomotor responses to the endothelins, expressed as the maximum contraction (Emax%), had the order ET-1 greater than ET-2 greater than ET-3 while the order of potency (pD2) was ET-1 = ET-2 greater than ET-3. ET-1 and ET-2 caused cross-desensitization, but did not affect the responses to ET-3. ET-3 did not cause cross-desensitization to ET-1 or ET-2 although it induced homologous desensitization. Finally, the effects of ET-1 and ET-2 were additive to those of ET-3. The additive effect of ET-3 to those of ET-1 or ET-2 was more difficult to demonstrate, given the profound contraction produced by ET-1 and to a lesser extent by ET-2. 3. In the trachea, the rank order of potency, additivity and desensitization were different from the pulmonary artery. Basically, all three peptides were equipotent but less potent than ET-1 in the artery. There was no evidence for additivity and only a slight tendency to tachyphylaxis was seen. 4. The guinea-pig pulmonary artery appears to be endowed with one receptor type which is sensitive to ET-1/ET-2 and with another receptor type which responds preferentially to ET-3. In the trachea, neither of these receptors appears to be present since all three peptides apparently act on a homogeneous population of receptors with characteristics different from those of the two arterial receptors. This suggests a third non-isopeptide selective type of endothelin receptor.


Adenosine receptors in rat basophilic leukaemia cells: transductional mechanisms and effects on 5-hydroxytryptamine release. 1. The presence of adenosine receptors linked to adenylate cyclase activity and their functional role in calcium-evoked 5-hydroxytryptamine (5-HT) release was investigated in rat basophilic leukaemia (RBL) cells, a widely used model for studying the molecular mechanisms responsible for stimulus-secretion coupling. 2. In [3H]-5-HT-loaded cells triggered to release by the calcium ionophore A23187, a biphasic modulation of 5-HT secretion was induced by adenosine analogues, with inhibition of stimulated release at nM and potentiation at microM concentrations, suggesting the presence of adenosine receptor subtypes mediating opposite effects on calcium-dependent release. This was also confirmed by results obtained with other agents interfering with adenosine pharmacology, such as adenosine deaminase and the non-selective A1/A2 antagonist 8-phenyl-theophylline. 3. Similar biphasic dose-response curves were obtained with a variety of adenosine analogues on basal adenylate cyclase activity in RBL cells, with inhibition and stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production at nM and microM concentrations, respectively. The rank order of potency of adenosine analogues for inhibition and stimulation of adenylate cyclase activity and the involvement of G-proteins in modulation of cyclic AMP levels suggested the presence of cyclase-linked A1 high-affinity and A2-like low-affinity adenosine receptor subtypes. However, the atypical antagonism profile displayed by adenosine receptor xanthine antagonists on cyclase stimulation suggested that the A2-like receptor expressed by RBL cells might represent a novel cyclase-coupled A2 receptor subtype.4. Micromolar concentrations of adenosine analogues could also increase inositol phospholipid hydrolysis and inositol tris-phosphate formation in both unstimulated cells and in cells triggered to release by the calcium ionophore. The stimulation was constant, small and additive to that exerted by the calcium ionophore.5. It is concluded that RBL cells express both A1 and A2-like adenosine receptors which exert opposite effects on 5-HT release and intracellular cyclic AMP levels. However, besides modulation of cyclic AMP levels, additional transduction pathways, such as modulation of phospholipase C activity, may contribute to the release response evoked by adenosine analogues in this cell-line.


Nedocromil sodium prevents in vivo generation of the eosinophilotactic substance induced by PAF but fails to antagonize its effects. 1 The intrathoracic injection of platelet activating factor (PAF) into rats induced a decrease in the pleural leucocyte numbers within 15 min, accompanied by a marked exudation, maximal 1 h later. After 6 h, concomitantly with the reduction of exudation, a marked increase in the number of mononuclear cells, neutrophils and eosinophils was observed. Within 24 h, the pleural eosinophil accumulation peaked and persisted up to 96 h. 2 Topical treatment with nedocromil sodium affected pleural exudation by PAF under conditions where systemic meclizine was ineffective. Nedocromil sodium blocked, dose-dependently, the increase in the pleural content of mononuclear cells, neutrophils and eosinophils, observed 6 h after PAF administration, as well as the eosinophilia 24 h later. Moreover, the co-incubation of peritoneal eosinophils with nedocromil sodium did not interfere with the migration triggered by PAF. 3 The transfer of the 6 h-PAF pleural washings from donor to recipient rats caused a selective pleural eosinophilia, which was clearly inhibited when nedocromil sodium was administered to donor, but not to recipient animals, showing that this drug interferes with the generation rather than with the expression of the eosinophilotactic activity(ies). 4 These findings indicate that the nedocromil sodium interferes with PAF-induced exudation and leucocyte accumulation, by a mechanism other than its ability to reduce the local effects of histamine and which may relate to suppression of the eosinophilotactic principle generation.


Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes. 1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.