Analysis of K+ transport by rabbit CCD: conductive pathways and K(+)-K+ exchange by Na(+)-K+ pump.

We studied the cellular pathways of K+ transport by the rabbit cortical collecting duct that was stimulated to absorb Na+ and to secrete K+. The vast majority of K+ secretion (into the lumen) was inhibited by benzamil, a blocker of epithelial Na+ channels. The residual K+ secretion was completely inhibited by ouabain. Thus all active K+ secretion was dependent on Na+ transport by the Na(+)-K+ pump. The passive pathways of K+ transport were further examined using tracer and electrophysiological measurements. K+ transfer across the apical membrane was predominantly or exclusively conductive; the apical K+ conductance was 31 mS/cm2. The basolateral membrane contained two pathways for K+ tracer translocation. The (barium-sensitive) conductive pathway accounted for a relatively small (12-20%) portion of the tracer permeation. A larger pathway appeared to be via K(+)-K+ exchange on the Na(+)-K+ pump. The magnitude of the Ba2(+)-sensitive (basolateral) K+ conductance predicted a substantially larger tracer flux than was actually measured. The best explanation for this difference is the presence of single-file diffusion through K+ channels on the apical and basolateral membranes. An analysis of the electrically silent K+ transport from lumen to bath suggests that the Na(+)-K+ pump can vary the ratio of its Na(+)-K+ and K(+)-K+ modes of operation. When the tubule is actively transporting Na+ and K+, the Na(+)-K+/K(+)-K+ turnover ratio is greater than 7. When Na+ transport is limited by inhibiting Na+ entry across the apical membrane, the ratio falls to less than 1. A major factor determining this ratio is probably the availability of Na+ to the cytoplasmic side of the pump.


Superoxide stimulates IP3-induced Ca2+ release from vascular smooth muscle sarcoplasmic reticulum.

Reactive oxygen intermediates (ROI) have been implicated in a variety of pathophysiological conditions, and vascular smooth muscle may be a site of damage in such oxygen toxicity. Mechanisms of the effects of these intermediates on vascular smooth muscle at the cellular level, however, have not been well studied. We have previously shown that xanthine oxidase (XO)-generated superoxide radicals (O2-.) inhibited the Ca(2+)-adenosine triphosphatase of vascular smooth muscle sarcoplasmic reticulum (SR) through mechanisms that do not involve H2O2 or hydroxyl radicals. In the present study, we report that the D-myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from bovine aortic SR was also affected by O2-(.). Hypoxanthine (100 microM) plus XO (10 mU/ml) in the presence of catalase (100 U/ml) stimulated the IP3-induced Ca2+ release from SR monitored using arsenazo III. At 10 microM IP3, the release was doubled by O2-. treatment. As a consequence of using the higher SR protein concentrations required to observe the Ca2+ release, this effect was independent of Ca2+ uptake inhibition induced by O2-(.). Since the effect of O2-. was not seen when a nonhydrolyzable analogue of IP3 was used to induce Ca2+ release, O-2. may be inhibiting the degradation processes of IP3.


Inhibitory effect of pindolol on hypoxic pulmonary vasoconstriction.

The effects of a beta-agonist (isoproterenol) and beta-antagonists (propranolol and pindolol) on hypoxic pulmonary vasoconstriction (HPV) and on changes in some chemical mediators were examined in 28 isolated blood-perfused dog lung lobes. Hypoxic challenge (4 min) was repeated twice, and each drug was administered at a bolus dose of 0.2 mg between the hypoxic periods. The first hypoxia increased pulmonary vascular resistance (PVR) by 33% or more in all groups. Both isoproterenol and pindolol inhibited the second HPV completely, but propranolol did not influence HPV. During normoxia, isoproterenol and pindolol significantly decreased PVR by 17.5 and 6.7% (P less than 0.01), respectively, but propranolol had no effect on PVR. In the control and propranolol groups, plasma levels of adenosine 3',5'-cyclic monophosphate (cAMP) significantly decreased during hypoxia, but those of prostaglandin E2, 6-ketoprostaglandin F1 alpha, and thromboxane B2 did not change. cAMP increased from 17.0 +/- 4.0 to 76.7 +/- 15.6 pmol/ml in the isoproterenol group (P less than 0.01), and prostaglandin E2 increased from 87.0 +/- 13.6 to 1,015.4 +/- 309.7 pg/ml in the pindolol group (P less than 0.05). The results suggest that the mechanism of HPV inhibition is different between isoproterenol and pindolol.


Oxygen radicals, enzymes, and fluid transport through pericardial interstitium.

The interstitium is the final link in the transportation of nutrients from the bloodstream to the individual cells of an organism. To assess interstitial fluid transport in normal and inflamed tissue, the hydration (H, ml H2O/g dry wt) and hydraulic conductivity (Kp, 10(-8) cm2.s-1.cmH2O-1) of bovine pericardial stroma were determined. The effect of enzymes and neutrophil-derived products of inflammation on the properties of the interstitial model were determined. Samples of the pericardium were exposed separately to trypsin, elastase, hyaluronidase, collagenase, superoxide radicals, and hydrogen peroxide. After exposure, the tissues were washed repeatedly in physiological saline and equilibrated in transport chambers heated to 37 degrees C and pressurized to 50 cmH2O. Fluid flow across the tissues was monitored. A section of tissue was removed and weighed. The tissue section was subsequently dried and reweighed. Tissue thickness, H, and Kp were calculated. H and Kp of the control tissues were 2.82 +/- 0.04 and 1.71 +/- 0.07, respectively. Hydration was significantly increased (22-38%) by exposure to trypsin, elastase, collagenase, and superoxide radicals. Kp increased significantly (30-1055%) in the groups treated with trypsin, hyaluronidase, collagenase, and superoxide radicals. The inflammatory mediators generally increased the hydration and/or the hydraulic conductivity of the model. These results indicate that neutrophil-derived products could be involved in the development of interstitial edema during the inflammatory process.


H2 receptor-mediated responses of aortic endothelial cells to histamine.

It is well known that umbilical vein endothelial cells express H1 receptors that mediate the various responses of these cells to histamine, including accumulation of inositol phosphates, rise of cytosolic Ca2+, increased permeability to macromolecules, and release of prostacyclin. In bovine aortic endothelial cells, histamine did not increase the level of inositol phosphates nor the release of prostacyclin. In contrast, it increased the adenosine 3',5'-cyclic monophosphate (cAMP) content of these cells. That response was obtained in the 1 to 100 microM range of concentrations and reached a maximum within 2 min of histamine addition. It was mimicked by the H2-specific agonist dimaprit, inhibited by the H2 antagonist ranitidine, and insensitive to the H1 antagonist mepyramine. Histamine reduced the permeability to albumin of bovine aortic endothelial cell monolayers; this paradoxical effect is likely to be mediated by the rise in cAMP, which is known to enhance the barrier property of the endothelium. In conclusion, bovine aortic endothelial cells are responsive to histamine, and this response is mediated by H2 and not H1 receptors.


Cardiovascular and renal actions of C-type natriuretic peptide.

Studies were performed in two groups of anesthetized dogs (n = 5 per group) to determine the cardiovascular and renal actions of synthetic C-type natriuretic peptide (CNP). Systemic infusion of CNP (group 1; 10 and 50 ng.kg-1.min-1 iv) resulted in marked cardiovascular hemodynamic effects characterized by a decrease in mean arterial pressure, cardiac output, and atrial pressures in association with a decrease in sodium excretion. Bolus administration of CNP (group 2; 5 micrograms/kg iv) to minimize cardiovascular hemodynamic changes resulted in only a transient decrease in arterial pressure. Sodium excretion decreased despite a return of arterial pressure to baseline. These biological responses were associated with increases in plasma guanosine 3',5'-cyclic monophosphate (cGMP) in both groups but with no change in urinary cGMP. With both systemic infusion or bolus administration of CNP, significant increases in plasma aldosterone were observed in association with increases in distal nephron sodium reabsorption. This study demonstrates that CNP exhibits profound systemic hemodynamic actions and is indirectly, or perhaps directly, antinatriuretic.


Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms.

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.


Ontogeny of cholinergic and adrenergic mechanisms in the frog (Rana temporaria) heart.

Histochemical techniques, field stimulation, and application of autonomic drugs were used to study neurotransmission in the heart during ontogenesis of Rana temporaria. Cholinesterase (ChE)-containing fibers, fluorescent chromaffinlike cells, and fluorescent fibers were found first in the heart at tadpole stages 40, 40, and 50, respectively. Inhibitory cholinergic and stimulatory adrenergic responses to field stimulation first appeared at stages 39-40 and 42, respectively. Inhibitory responses to acetylcholine (ACh) and stimulatory responses to epinephrine (Epi) were observed as early as stages 31 and 32. The concentrations producing half-maximal response values for both neurotransmitters increased during development. Indirect evidence was obtained that the subsensitivity of tadpole hearts to ACh was due to increased hydrolysis of ACh by tissue ChE and that the subsensitivity of adult frog heart to Epi could be connected with a maturation of the neuronal uptake mechanism. The pA2 values for atropine and propranolol were 10 times greater in tadpoles than in adults. The main conclusion is that the cholino- and adrenoreactive systems appear in the frog heart cells before they become innervated and the sensitivity of these systems to neurotransmitters does not increase with innervation.


The role of B1- and B2-kinin receptors in the renal tubular and hemodynamic response to bradykinin.

Bradykinin (BK) is known to induce diuresis (UV), natriuresis (UNaV), and increased renal blood flow (RPF) with little or no change in glomerular filtration rate (GFR). In this study, BK is infused alone and concurrently with B1- or B2-kinin receptor antagonists into the left kidney of pentobarbital-anesthetized dogs. The intrarenal infusion of BK (bolus: 0.5 microgram/kg, followed by a sustaining dose: 0.05 micrograms.kg-1.min-1) affected left kidney function only. In the left kidney, UV increased from 0.42 +/- 0.21 to a maximum of 1.88 +/- 0.55 ml/min (P less than 0.01) and UNaV rose from 55 +/- 13 to 160 +/- 17 mueq/min (P less than 0.01), while RPF was enhanced from 86 +/- 11 to 125 +/- 24 ml/min (P less than 0.05), and GFR remained unchanged. When a B1-receptor antagonist ([Leu8]-des-Arg9-BK; 2.0 micrograms.kg-1.min-1) was infused concurrently with BK, the increase in urine flow was not different from BK alone. UNaV was transiently attenuated by 50% in this group (P less than 0.05). A B2-receptor antagonist (D-Arg0,[Hyp3,D-Phe7]-BK; 2.0 micrograms.kg-1.min-1) infused with BK significantly (P less than 0.05) and selectively inhibited by 50% the maximal diuresis provoked by BK alone. UNaV in this group was not different from that induced by BK alone. Finally, the concurrent infusion of either B1- or B2-antagonist completely inhibited the rise in RPF observed when BK was infused alone. We conclude that BK infused into the renal artery of dogs in vivo can alter UV and UNaV independently of global renal hemodynamic (RPF and GFR) changes.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin-1 potentiation of angiotensin II stimulation of aldosterone production.

Endothelin-1 (ET-1) binds to specific receptors in cultured bovine adrenal glomerulosa cells and stimulates aldosterone secretion with a 50% effective concentration (EC50) of 300 +/- 80 pM (mean +/- SE). The relative stimulatory potency for ET-1 is significantly less than that of angiotensin II (ANG II). The incubation of calf zona glomerulosa cells in primary culture with ET-1 and ANG II resulted in a significant potentiation of ANG II effect on aldosterone secretion. The EC50 of ET-1 potentiation of ANG II-induced stimulation of aldosterone secretion was 40 +/- 5 pM (mean +/- SE, n = 4), which is lower than the EC50 for ET-1 stimulation of aldosterone secretion. Adrenocorticotropic hormone (ACTH) stimulation of aldosterone secretion, but not that of potassium, was also potentiated by ET-1, but to a lesser degree. ET-1 and ET-1-mediated potentiation of ANG II-stimulated aldosterone biosynthesis increased both the early and late pathways of aldosterone biosynthesis, but the potentiation was greater for the early pathway. Preincubation with ET-1 for at least 15 min, followed by extensive washing to remove bound ET-1, also resulted in persistent potentiation of ANG II-mediated aldosterone secretion. ET-2, sarafotoxin, and vasoactive intestinal contractor potentiation of ANG II action were very similar to that of ET-1. ET-3 and Big-ET-1 potentiated ANG II stimulation only at the highest doses tested and the proendothelin-(110-130) fragment was inactive. ET-1 potentiation of ANG II action is likely to be mediated through an ETB receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistochemical identification of aggregated actin filaments in formalin-fixed, paraffin-embedded sections. I. A study of infantile digital fibromatosis by a new pretreatment.

Some authors have claimed that actin is not immunostained in characteristic intracytoplasmic inclusions of infantile digital fibromatosis, whereas others have claimed that it is. Formalin-fixed specimens were used in the former studies; specimens fixed in alcohol used in the latter studies. Actin at other sites, such as the rim of the inclusions, was distinctly immunostained even in the formalin-fixed specimens. Such phenomena make it difficult to accept the loss of antigenicity of actin as a result of formalin fixation. The use of usual pretreatment with trypsin provided the same results. We were able to immunostain actin distinctly and strongly in the inclusions for the first time in formalin-fixed specimens by combining KOH in 70% ethanol and trypsin. This successful staining results from the adequate etching effect of trypsin, which occurs because of a loosening of proteins in the inclusions due to KOH. These phenomena suggest that steric hindrance of antigen determinant has occurred only in the inclusions as a result of intramolecular cross-linkage, because of extremely dense accumulation of actin filaments in the inclusions.


Frequency of Epstein-Barr viral DNA in "Western" sinonasal and Waldeyer's ring non-Hodgkin's lymphomas.

Sinonasal non-Hodgkin's lymphomas (SNHL) of B- or T-cell immunophenotype have been associated with the Epstein-Barr virus (EBV) in Asian patients. We investigated eight sinonasal and 10 Waldeyer's ring NHL from Western patients for evidence of EBV genomes using a sensitive in situ hybridization technique. EBV DNA was detected in each of three sinonasal NHL with a T-cell immunophenotype and two of five cases with a B-cell immunophenotype. Two of 10 B-cell Waldeyer's ring NHL were positive for EBV genomes. In each positive case, EBV genomes were evenly distributed among the neoplastic cells, whereas no evidence of EBV in associated nonneoplastic lymphocytes or epithelium was seen. The results indicate that B-cell and T-cell sinonasal NHL are associated with EBV in Western as well as in Asian patients, and that EBV may have a role in oncogenesis in NHL of the upper aerodigestive tract. The strong association of EBV with nasopharyngeal carcinoma suggests that the anatomic site is important in the development of EBV-related neoplasms.


Meal-induced jejunal absorption requires intact neural pathways.

A signal for meal-induced absorption originates from the small intestine and is transmitted to a luminally excluded segment of the proximal jejunum (Thiry-Vella [TV] fistula). Using intraluminal topical anesthesia with oxethazaine, this study assessed the role of intestinal neural pathways in basal and postprandial jejunal water and electrolyte absorption. Studies (n = 45) were performed on dogs with 25-cm proximal jejunal TV fistulae and feeding jejunostomies, using luminal perfusion with 14C-polyethylene glycol. The animals were randomized into five study groups: (1) jejunostomy oxethazaine alone, (2) jejunostomy water and jejunal meal, (3) jejunostomy oxethazaine and jejunal meal, (4) TV fistula water and jejunal meal, and (5) TV fistula oxethazaine and jejunal meal. The jejunal meal significantly increased TV fistula absorption, whereas oxethazaine significantly reduced basal absorption when administered via the TV fistula and postprandial absorption when administered via the jejunostomy (p less than 0.05). TV fistula oxethazaine did not diminish the magnitude of postprandial absorption. We conclude that intact intestinal neurotransmission is necessary for maintenance of the normal basal absorptive state of the proximal jejunum and for the generation of a normal meal-stimulated proabsorptive signal from the small intestine. A nonneural mechanism appears to be of predominant importance in transmitting the proabsorptive signal from the intact gastrointestinal tract to the TV fistula.


Herpes simplex hepatitis before and after acyclovir treatment. Immunohistochemical and in situ hybridization study.

A healthy 20-year-old woman developed herpes simplex virus (HSV) hepatitis. The diagnosis was made by needle biopsy of the liver, and the patient was intravenously treated with acyclovir for 15 consecutive days (total dose, 21 g). The liver biopsy specimen and liver tissue obtained at autopsy were processed for immunoperoxidase staining with rabbit anti-HSV and for DNA-DNA in situ hybridization. The liver biopsy tissue revealed massive necrosis of hepatocytes, which were strongly positive for HSV with both immunoperoxidase and in situ hybridization methods. The liver tissue obtained at autopsy showed regenerative nodules of hepatocytes, surrounded by connective tissue stroma. Within the connective tissue there were completely necrotic hepatocytes, which were positive for HSV with the immunoperoxidase method but almost completely negative with the in situ hybridization method, except for a very few HSV DNA-positive hepatocytic nuclei. It was concluded that immunoperoxidase staining with anti-HSV is a sensitive method with which to detect ongoing and previous HSV infection, whereas the in situ hybridization method is specific for HSV-DNA from viable HSV.


Female adnexal tumor of wolffian origin.

We describe a female adnexal tumor of probable wolffian origin with stage 2B disease. The presence of peritoneal implants suggests a more aggressive clinical course than is usually expected. We present the findings of light microscopic and ultrastructural evaluation as well as those of immunohistochemical and DNA ploidy analysis, which, to our knowledge, have not been previously described for this type of tumor.


Giant cystosarcoma phyllodes of the prostate associated with adenocarcinoma.

Cystosarcoma phyllodes of the prostate is a rare neoplasm, occurring in adult men. It closely resembles the not uncommon tumor of the female breast and usually behaves in a similar manner. This case of benign cystosarcoma phyllodes of the prostate occurred in a 53-year-old man who presented with increasing abdominal girth and underwent exploratory laparotomy and removal of the 11.2-kg tumor. It was remarkable for its very large size and the presence of foci of well-differentiated adenocarcinoma, prostatic acinar type. The glandular epithelium of both the phyllodes tumor and the carcinoma were immunoreactive for cytokeratin, epithelial membrane antigen, prostate-specific antigen, and prostate-specific acid phosphatase. The presence of typical prostatic type adenocarcinoma and this immunoreactivity pattern strongly supports a prostatic origin for this rare neoplasm.


Explanation at the opioid receptor level for differing toxicity of morphine and morphine 6-glucuronide.

The radiolabelled opioid receptor binding affinities of morphine and its active metabolite morphine 6-glucuronide at the total mu, mu 1, mu 2 and delta receptors were determined. Morphine 6-glucuronide was found to have a 4-fold lower affinity for the mu 2 receptor (IC50 17 nM and 82 nM for morphine and morphine 6-glucuronide respectively, P = 0.01), the receptor postulated to be responsible for mediating the respiratory depression and gastrointestinal effects after morphine. This provides a possible explanation for the reduced respiratory depression and vomiting seen following morphine 6-glucuronide in man. A similar reduction in affinity of morphine 6-glucuronide was seen at the total mu receptor whilst there was no significant difference seen at the mu 1 or delta receptor. Hence the increased analgesic potency of morphine 6-glucuronide over morphine remains unexplained.


Chemotherapy of non-small cell lung carcinoma guided by an in vitro drug resistance assay measuring total tumour cell kill.

Specimens from 45 patients with previously-untreated non-small cell lung cancer (NSCLC) were tested for in vitro chemosensitivity to ten drugs utilising the DiSC assay, which measures cell kill in the total (largely non-dividing) tumour cell population. Thirty-five assays were successful and 25 patients with advanced disease subsequently received chemotherapy with the 'best' three drugs selected by the assay. Six patients were Karnofsky performance status 60 or less and the median pretreatment weight loss was 8.5%. Nine patients had a partial response (response rate = 36%; 95% confidence interval = 17-55%) and the median survival of all patients was 202 days. Specimens from responding patients were significantly more sensitive in the assay to drugs in general (especially to etoposide and to 'natural product' drugs) and to the drugs used in treatment than were specimens from non-responding patients. In vitro drug resistance differences between responding and non-responding patients were of greater significance than were differences between other clinical and laboratory measurements. Assay results classified patients into two cohorts, having relatively high and low probabilities of responding to chemotherapy. Assay results also identified patient cohorts with above average and below average durations of survival. Five patients (20%) were found to have tumours with extreme drug resistance (EDR), defined as assay results for the average of all ten tested drugs falling greater than one standard deviation more resistant than the median for all tumours assayed, and none of these patients with EDR responded to chemotherapy.


Overexpression of the p53 protein and allele loss at 17p13 in ovarian carcinoma.

Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours (16 serous adenocarcinomas, six endometrioid carcinomas, one mucinous adenocarcinoma, one mucinous borderline tumour and three benign adenomas). Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance.


Manipulations of the renin-angiotensin system and intake of a sweetened alcoholic beverage among rats.

Standard laboratory rats were maintained on a daily regimen involving deprivation of fluids for 22 h followed by a 2-h opportunity to drink water and a sweetened alcoholic beverage. Angiotensin II, in doses ranging from 0.1 to 1.25 mg/kg, dose relatedly decreased rats' mean intake of ethanol. All doses increased rats' mean intake of water. Angiotensin II, 0.25 mg/kg, reliably reduced intake of ethanol when it was presented alone during the 1st h of the daily 2-h drinking session, and reliably increased intake of water when it was subsequently presented alone during the 2nd h. Thus the reduction in intake of ethanol seen when the alcoholic beverage is presented concurrently with water is probably not merely due to the increase in intake of water. Lisinopril, an angiotensin converting enzyme inhibitor, in doses of 0.3, 1.0, and 3.0 mg/kg, dose relatedly decreased intake of ethanol, but only after several days of injections. Concurrent intake of water was increased dose relatedly. When injections of lisinopril ceased, intakes of both ethanol and water took several days to return to control levels. Pretreatment with lisinopril, 3.0 mg/kg, for 8 days, had no effect on subsequent intakes of either water or ethanol. Lisinopril, 3.0 mg/kg, had no effect on rats' intake of a sweet solution without ethanol. These results confirm previous work and extend the data base supporting the idea that the renin-angiotensin system plays a role in modulating intake of ethanol.


Three types of human lung tumour cell lines can be distinguished according to surface expression of endogenous urokinase and their capacity to bind exogenous urokinase.

This study evaluates the cell surface expression of urokinase-type plasminogen activator (u-PA) and the capacity to bind exogenous urokinase as possible parameters for the distinction of various types of human lung tumours. Twelve different tumour cell lines including four small cell carcinoma, two large cell carcinoma, three squamous cell carcinoma, one adenocarcinoma and two mesothelioma cell lines of lung origin were investigated. Surface expression of endogenous u-PA was determined in a cellular radioimmunoassay (CRIA) using the u-PA-specific monoclonal antibody 98/6. To estimate additional u-PA binding capacity, exogenous two-chain, 54 kDa u-PA was employed in the CRIA. The influence of phorbol ester (PMA) treatment on expression and binding of these molecules was studied. Three different groups of lung tumour cell lines could be distinguished according to their expression of u-PA and u-PA-binding ability: (i) non small cell lung carcinoma (NSCLC) cell lines of squamous cell carcinoma/adenocarcinoma origin expressed small amounts of u-PA and bound little u-PA. Large cell carcinoma cell lines expressed high amounts of u-PA and bound large amounts of u-PA. In general, expression of u-PA and u-PA binding was enhanced after PMA treatment. (ii) Mesothelioma cell lines did not express u-PA, but were able to bind u-PA. (iii) Small cell carcinoma (SCLC) lines were devoid of surface-expressed u-PA and could not bind u-PA, both under untreated and PMA-treated conditions. It could thus be demonstrated that these three groups of lung tumour cell lines differ in their ability to express u-PA and to bind external u-PA. This may reflect the different in vivo growth behaviour and origin of the respective tumour groups.


Non-invasive assessment of human tumour hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study.

Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 (primary small cell lung carcinoma), 1.9 (primary malignant fibrous histiocytoma) and 3.2 (brain metastasis from small cell lung carcinoma) at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.


Relation between exposure to respirable silica dust and silicosis in a tungsten mine in China.

To estimate the quantitative relation between exposure to respirable silica dust and risk of an attack of silicosis, 1151 workers exposed to silica dust and employed from 1958 to 1987 in a tungsten mine in China were investigated. The results showed that the ratio of respirable silica dust concentration to total silica dust concentration was 0.529. Then, the total silica dust concentration in historical surveillance and monitoring data was converted to respirable silica dust concentration. The free silica content in respirable dust determined by x ray diffraction averaged 24.7%. Multiple logistic regression was used for the dichotomous dependent variables (presence or absence of silicosis). The independent variables in the multiple logistic regression with presence of silicosis as the dependent variable were age when first exposed, tuberculosis (presence or absence), and cumulative exposure to respirable silica dust. The partial regression coefficient of individual cumulative exposure was estimated as 0.079. It implied a positive association between exposure to respirable silica dust and risk of an attack of silicosis. The exposure limit for respirable silica dust was estimated as 0.24 mg/m3 under given conditions.


Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B.

Activation of maturation-promoting factor at the onset of mitosis requires the tyrosine dephosphorylation of one of its components, the cdc2 protein kinase. cdc25 is the specific tyrosine phosphatase that activates cdc2. We find that Xenopus oocytes contain a relative of cdc25, p72. In Xenopus embryos the abundance of p72 does not oscillate during the cell cycle. However, p72 directly associates with cdc2-cyclin B in a cell cycle-dependent manner, reaching a peak at M phase. The M phase kinase that associates with p72 is catalytically active. These results suggest that the mechanism by which cdc25 triggers cdc2 activation involves a periodic physical association between cdc25 and the cyclin B-cdc2 complex and also that mitotic control can be affected by mechanisms other than transcriptional regulation of the cdc25 gene.


Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum.

Resident luminal endoplasmic reticulum (ER) proteins carry a targeting signal (usually KDEL in animal cells) that allows their retrieval from later stages of the secretory pathway. In yeast, the receptor that promotes this selective retrograde transport has been identified as the product of the ERD2 gene. We describe here the properties of a human homolog of this protein (hERD2). Overproduction of hERD2 improves retention of a protein with a weakly recognized variant signal (DDEL). Moreover, overexpression of KDEL or DDEL ligands causes a redistribution of hERD2 from the Golgi apparatus to the ER. Mutation of hERD2 alters the ligand specificity of this effect, implying that it interacts directly with the retained proteins. Ligand control of receptor movement may limit retrograde flow and thus minimize fruitless recycling of secretory proteins.


Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.


9-cis retinoic acid is a high affinity ligand for the retinoid X receptor.

All-trans retinoic acid (RA) has previously been shown to modulate the transcriptional properties of the retinoic acid receptor (RAR) and retinoid X receptor (RXR). The inability of all-trans RA to bind to RXR suggests that it may be metabolized to a more active high affinity ligand. We report here an experimental approach that has identified 9-cis RA as an RXR ligand. It is up to 40-fold more potent than all-trans RA in transfection assays and binds with high affinity. The production of 9-cis RA in cultured cells and the identification of this molecule in liver and kidney demonstrates the existence of this molecule in living organisms. The discovery of this novel hormone points to the key role retinoid metabolism may have in generating new signaling pathways.


A diacylglycerol kinase is involved in the regulation of interleukin-2 synthesis in Jurkat T cells.

Diacylglycerol (DAG) plays a prominent role in several activation processes because of its ability, in the presence of calcium ions and phosphatidylserine, to activate C kinase. In T lymphocytes, however, DAG, produced in response to activating mAb or lectins, is rapidly metabolized into phosphatidic acid (PA) which may participate in further steps of activation. We thus investigated the involvement of a DAG kinase in several events subsequent to the activation of Jurkat T cells by CD3 mAb or phytohemagglutinin (PHA). We showed that three inhibitors of DAG kinase abrogated PA production and impaired calcium release from intracellular compartment, restored phosphatidylserine synthesis, and finally blocked IL-2 production in CD3- and PHA-stimulated cells. Postactivation DAG levels were not modified and DAG kinase inhibitors lowered IL-2 secretion even in the presence of phorbol ester that directly activates the C kinase. These results clearly demonstrate that, beside the effect of DAG on C kinase, DAG kinase dependent production of PA is crucial for further steps of T cell activation.


Impairment of macrophage activation and granuloma formation by protein deprivation in mice.

Protein-calorie malnutrition predisposes to infection by intracellular pathogens, but the basis for this predisposition is unclear. We studied the effect of protein deprivation on mouse peritoneal macrophage function and on granuloma formation during infection by bacille Calmette-Gueŕin (BCG). Injection of lipopolysaccharide (LPS) to induce inflammation elicited fewer peritoneal cells from mice fed a 2.5% protein diet than from mice fed an isocaloric chow in which protein calories constituted 24% of the total. LPS-elicited macrophages from protein-deprived mice demonstrated a reduction in spreading, total cell protein, cell lactate dehydrogenase, and release of superoxide anion (O2-) in response to stimulation. Priming in vitro by interferon (IFN)-gamma for enhanced release of O2- was also significantly impaired in protein-deprived mice. This defect was reversible by repletion with 24% protein diet for 10 days. Impairment of macrophage function in protein-deprived mice was further evidenced by an impaired capacity to express Ia antigen in response to IFN-gamma and by reduced production of IL-1 activity in response to LPS. Infection by BCG in protein-deprived mice was characterized by impaired granuloma development in liver, lungs, and spleen. Thus, in this model, protein deprivation significantly impaired macrophage activation, as assessed by morphologic, metabolic, and functional criteria. This impairment might compromise immune effector mechanisms dependent on macrophage activation, including rejection of intracellular pathogens.


Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.


Hydroxyl radical-induced characteristic chemiluminescent spectra from plasma of hemodialysis patients.

Plasma from hemodialysis patients evoked weak photon emissions (chemiluminescence) in a characteristic emission spectrum with a peak at 430 nm, attributed to attack by hydroxyl radicals generated from the iron-catalyzed breakdown of hydrogen peroxide (Fenton reaction), whereas plasma from normal healthy subjects showed a rather weak red chemiluminescence peak at around 680 nm, similar to that resulting from attack by hydroxyl radicals. However, the addition of hydrogen peroxide in the absence of divalent irons induced almost the same red chemiluminescent emission spectrum in both plasmas. The HPLC-gel-filtration chromatography carried out with both plasmas revealed that a primary emitter evoking a peak emission at 430 nm was located in the fraction of lower-molecular-mass substances in fractionated plasma from hemodialysis patients. In contrast, the elution peaks evoking red chemiluminescence with the addition of hydrogen peroxide were mainly observed for the higher-molecular-mass fraction, as determined by gel chromatography of both plasmas. Therefore, the observation of a chemiluminescence peak at 430 nm, induced by the generation of hydroxyl radicals, correlated well with chemiluminescent emissions in plasma samples from patients with chronic renal failure. Spectral analyses of clinical samples that show weak chemiluminescence by forced oxidation by such an active oxygen may provide a new and more sensitive method for diagnosing metabolic disorders.


Serum steroid hormonal profiles by reversed-phase liquid chromatography in patients with 17-hydroxylase deficiency and in an affected family.

We used an optimized isocratic reversed-phase high-performance liquid-chromatographic procedure to separate and measure 12 steroid hormones, and studied the steroid hormone profiles in sera from three patients with 17-hydroxylase deficiency (17-OHD). Two of the patients were sisters, one of whom (II-3), expressing normotension and primary amenorrhea, was diagnosed on the basis of chromatographic data and followed up for seven years. The untreated patients had obvious abnormalities on chromatograms of serum extracts, characterized by markedly increased corticosterone (B) and decreased or undetectable cortisol (F) and cortisone (E). The concentration of 11-deoxycorticosterone was much greater in the patient with classical symptoms than in the normotensive patient. In all three patients, concentrations of aldosterone were within the normal range, but concentrations of progesterone were much lower than in the patients with 21-hydroxylase deficiency. We evaluated the responses to corticotropin and dexamethasone. HPLC evaluation of the serum steroid profiles before and after corticotropin stimulation in the affected family showed that in the parents and one other sibling, concentrations of F before and after stimulation were within the normal ranges. The sums of the ratio of B to F before and the ratio of B to F after corticotropin stimulation (sigma B/F) in the parents and the other sibling were 0.292, 0.496, and 0.614, respectively, all much higher than the normal value (mean +/- SD: 0.164 +/- 0.038). Thus the sigma B/F value may be a hormonal marker of heterozygotes carrying this defect.


Eccrine acrospiroma of the external auditory canal.

Tumors of the external auditory canal include osteomas, ceruminomas, sebaceous cysts, keloids, basal cell carcinomas, squamous cell carcinomas, and, in rare cases, eccrine acrospiromas. We describe an eccrine acrospiroma arising from the distal external auditory canal, which was associated with decreased hearing on the affected side. Surgical resection of the tumor resulted in improved hearing.


Familial adenomatous polyposis: results following ileal pouch-anal anastomosis and ileorectostomy.

To compare the clinical and functional results of ileorectostomy (IR) and ileal pouch-anal anastomosis (IPAA) in patients with familial adenomatous polyposis (FAP), we reviewed the results of 94 IPAA patients and 21 IR patients who were operated upon between 1978 and 1988. The groups were similar with respect to age and sex. None of the patients died postoperatively. Postoperative complications occurred in 28 percent of the IPAA group and in 17 percent of the IR group (P greater than 0.1). Seven percent of IPAA patients described symptoms compatible with pouchitis. Sixty-one percent of IR patients required subsequent fulguration of rectal polyps at least once. IR patients had a mean (+/- SD) of 4 (+/- 2) stools per day, while IPAA patients had 5 (+/- 2) stools per day (P greater than 0.05). No significant difference in daytime soiling was present between IR (6 percent) and IPAA (4 percent). Nighttime spotting was also similar between the two groups. Nighttime soiling, however, was reported by 4 percent of IPAA patients but not by IR patients (P less than 0.05). One IPAA patient (1 percent) required pouch excision for a desmoid tumor, while two IR patients (11 percent) required proctectomy and ileostomy for recurrent dysplastic polyps (P less than 0.05). Adhesions and a shortened ileal mesentery prevented the construction of an ileoanal procedure in these latter patients. In conclusion, the postoperative complication rate and functional results are similar after IR and IPAA in patients with FAP; however, IR does not eradicate rectal polyps and may indeed preclude IPAA for those requiring subsequent proctectomy.


Noncytotoxic drug therapy for intra-abdominal desmoid tumor in patients with familial adenomatous polyposis.

Forty of 416 patients with familial adenomatous polyposis were noted to have intra-abdominal desmoid tumors, and a subgroup of 16 were treated with noncytotoxic drug therapy. Drugs used were sulindac (14 patients), sulindac plus tamoxifen (3 patients), indomethacin (4 patients), tamoxifen (4 patients), progesterone (DEPO-PROVERA; Upjohn Co., Kalamazoo, MI) (2 patients), and testolactone (1 patient). Therapy with these drugs for continuous periods of six months or more resulted in three complete and seven partial remissions. When treated patients were compared with untreated patients (n = 12), there were significant benefits for the treated group, both in reduction of desmoid size and in improvement of symptoms, despite the inherent selection bias against this. Sulindac was the only drug used in enough patients to permit independent evaluation of its effect, with one complete and seven partial reductions of tumor size. Some patients had a delayed response to sulindac, with tumor shrinkage occurring after an initial period of tumor enlargement. When using sulindac for the treatment of desmoid tumors, this phenomenon should be considered.


Highly malignant carcinoma in chronic ulcerative colitis without preceding dysplasia or DNA aneuploidy. Report of a case.

A 41-year-old female with a history of total ulcerative colitis for 15 years is presented. After eight years, she was enrolled in a colonoscopic surveillance program with regular examinations every second year and with biopsy sampling for histologic assessment of dysplasia as well as for flow cytometric analysis. Neither dysplasia nor DNA aneupoloidy developed during the course of the follow-up, but, after seven years, the patient developed a rapidly growing malignant stricture in the lower rectum. At the time of diagnosis, a local gluteal metastasis was found. Following preoperative radiation therapy, laparotomy disclosed a rectal cancer with local growth in the pelvis. Despite an attempt to perform curative surgery, the patient deteriorated and died within four months after the diagnosis. The carcinoma was of a poorly differentiated, mucinous, signet ring cell type, and DNA analyses of both the tumor and its metastases were diploid. Retrospective analyses of mucin content in colonoscopic biopsies showed a gradual shift from sulfated mucin to sialomucin. This case underlines the fact that even rigorous follow-ups offer no absolute guarantee against incurable malignancy in surveillance programs for ulcerative colitis despite the inclusion of DNA analyses.


Effects of neutrophils in rat luteal cells.

Ovarian function may be modulated by cells of the immune system. We have investigated the role of neutrophils (polymorphonuclear leukocytes) on rat luteal cell function. Activated neutrophils inhibited LH-sensitive cAMP accumulation, which was dependent on neutrophil cell number. At a concentration of 10(6) neutrophils/ml and 10(5) luteal cells/ml, LH-stimulated cAMP accumulation was inhibited by 50%. The inhibitory effect of activated neutrophils was reversed by superoxide dismutase (SOD) and catalase. LH-stimulated progesterone production was also inhibited by activated neutrophils. Progesterone production by 10(5) luteal cells was inhibited approximately 20% in the presence of 10(6) activated neutrophils, and this inhibition was blocked by SOD and catalase. Conditioned medium from activated neutrophils also produced inhibitory effects on LH-stimulated cAMP accumulation and progesterone production, which could be reversed by SOD and catalase. The phosphodiesterase inhibitor isobutylmethylxanthine had no significant effect on the inhibition of cAMP accumulation by conditioned medium from activated neutrophils. Luteal cells loaded with a fluorescent indicator for determining intracellular reactive oxygen species (dichlorofluorescein diacetate) showed increased fluorescence in the presence of activated neutrophils. No increase in fluorescence occurred in the absence of neutrophils or in the presence of SOD and catalase. These studies demonstrate that reactive oxygen species produced by activated neutrophils can enter the luteal cell and cause antigonadotropic effects. Although the experimental model used in the present studies may not be truly physiological, the data demonstrate that neutrophils may play a role in functional and structural regression of the corpus luteum in the rat.


Calcium: its role in alpha 1-adrenergic stimulation of atrial natriuretic peptide secretion.

alpha 1-Adrenergic agonists increase atrial natriuretic peptide (ANP) secretion. The mechanism of alpha 1-adrenergic-stimulated secretion is not known. In this study we examine the calcium dependency of alpha 1-agonist-stimulated ANP secretion. Isolated superfused rat left atria paced at 2 Hz were used for study. Superfusion with 10 microM phenylephrine increased ANP secretion by 2-fold. Lowering the superfusate calcium concentration from 1.8 to 0.2 mM totally negated the secretory response to phenylephrine. To determine whether this reflected a reduction in calcium influx, reduced calcium release from the sarcoplasmic reticulum (SR), or both, atria were superfused with 1 microM ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on phenylephrine-stimulated ANP secretion. Atria were superfused with 10 microM nitrendipine to determine whether calcium influx through voltage-dependent calcium channels was a mechanism of calcium entry for stimulation. Nitrendipine inhibited phenylephrine-stimulated ANP secretion by 49% without interfering with alpha 1-adrenergic antagonist receptor binding. This finding was supported by the observation that phenylephrine-stimulated secretion was 52% lower in nonbeating atria. alpha 1-Adrenergic agonists have been reported to enhance Na-H antiporter activity. To determine whether the resulting rise in intracellular sodium may alter Na-Ca exchange to raise intracellular calcium levels, atria were superfused with the Na-H antiporter inhibitor, 5-(N,N-hexamethylene)amiloride. Superfusion with 25 microM 5-(N,N-hexamethylene)amiloride did not inhibit phenylephrine-stimulated ANP secretion. Lastly, the calcium dependency of the maintenance of an established response to phenylephrine was examined. Atria were superfused with phenylephrine in buffer containing 1.8 mM calcium for 45 min, followed by superfusion with phenylephrine in 0.2 mM calcium for 30 min. There was no fall in phenylephrine-stimulated secretion by atria superfused in 0.2 mM calcium. In contrast, addition of the alpha 1-adrenergic antagonist phentolamine induced an immediate fall in phenylephrine-stimulated ANP secretion. We conclude that 1) calcium influx is necessary to initiate alpha 1-agonist-stimulated ANP secretion; 2) calcium release from the SR does not play a role in alpha 1-agonist-stimulated secretion; 3) calcium entry through L-type calcium channels is responsible for half of the calcium influx; 4) enhanced Na-H antiporter activity does not play a role in alpha 1-agonist-stimulated secretion; and 5) maintenance of alpha 1-agonist-stimulated secretion is not dependent on calcium influx.


Rat melanin-concentrating hormone stimulates adrenocorticotropin secretion: evidence for a site of action in brain regions protected by the blood-brain barrier.

Melanin-concentrating hormone (MCH) is a peptide reported to inhibit ACTH and cortisol secretion in teleost fish. Its ability to modify the activity of rat corticotrophs, however, remains controversial. We report here that while the peripheral injection of rat (r) MCH failed to alter plasma ACTH levels of conscious rats with an intact blood-brain barrier (BBB), it significantly activated the hypothalamic-pituitary-adrenocortical axis of rats with increased BBB permeability induced by protamine sulfate administration into the internal carotid artery. Similarly, the intracerebroventricular injection of this peptide into rats with intact BBB measurably released ACTH. The ACTH response to rMCH was markedly, but not totally, inhibited by passive immunoneutralization of CRF. These results indicate that rMCH acts within the central nervous system to stimulate the hypothalamic-pituitary-adrenocortical axis of rats, and that the site of action of the peptide is located in brain structures protected by the BBB. Activation of CRF-secreting neurons represents an important final pathway, although other regulatory factors also seem to be involved.


The placental lactogen receptor in maternal and fetal sheep liver: regulation by glucose and role in the pathogenesis of fasting during pregnancy.

To clarify the roles of glucose and insulin in the regulation of the PL receptor in fetal and maternal sheep liver, we administered iv glucose to pregnant ewes during a 72-h fast. The binding of ovine PL (oPL) to hepatic membranes from glucose-infused ewes and their fetuses was compared with the binding of oPL to tissues of fasted, saline-infused sheep and sheep fed normally ad libitum. Fasting of pregnant ewes caused a 58-70% reduction in the number of PL receptors in fetal and maternal liver. Intravenous administration of glucose during fasting increased the number of PL receptors in fetal liver by 137.4%. In contrast, glucose administration during fasting had no effect on the number of PL receptors in maternal liver. The number of PL binding sites in fetal liver correlated positively with fetal weight (r = 0.59) and length (r = 0.54) and with fetal plasma glucose (r = 0.69) and insulin (r = 0.55) concentrations. In contrast, PL binding was inversely related to fetal plasma oPL concentrations (r = -0.70). These findings suggested that glucose, insulin, and/or oPL may regulate PL binding in the ovine fetus. To determine whether glucose or insulin exert direct effects on the PL receptor in ovine fetal tissues, we examined the binding of radiolabeled oPL to ovine fetal hepatocytes and fibroblasts in culture. The specific binding of oPL to fetal hepatocytes was low and variable (1.0 +/- 0.5%) and it was not possible to assess reliably the effects of glucose or insulin supplementation. The specific binding of oPL to fetal fibroblasts (5.4 +/- 0.6%/mg) was unaffected by variations in media glucose concentrations (5.5-16.5 mM) or by pretreatment with insulin (10-1000 ng/ml). The results of these studies demonstrate that glucose and other nutritional factors regulate the expression of the PL receptor in fetal and maternal sheep liver. Alterations in PL binding play roles in the metabolic adaptation of the mother and fetus to nutritional deprivation and stress.


12-O-tetradecanoylphorbol-13-acetate inhibits osteoclast-like cell differentiation in rat bone marrow cultures by inducing macrophage polykaryons.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10(-9) M stimulated the formation of osteoclast-like multinucleated cells (MNCs) in the presence of 1 alpha,25-dihydroxyvitamin D3 in rat bone marrow cultures. However, at 10(-7) M, it clearly inhibited 1 alpha,25-dihydroxyvitamin D3-dependent osteoclast-like MNC formation at 6 days of culture. In cultures treated with 10(-7) M TPA, numerous MNCs that lack the marker enzyme tartrate-resistant acid phosphatase (TRAP) were formed. These TRAP-negative MNCs had neither receptors for calcitonin nor dentine-resorbing activity. The reactivity of the cells against antirat macrophage antibodies was completely different from that of authentic osteoclasts. These data suggest that TRAP-negative MNCs formed in the presence of 10(-7) M TPA are macrophage polykaryons. Time-course studies showed that 10(-7) M TPA stimulated osteoclast-like MNC formation at 4 days of culture, but these osteoclast-like MNCs were converted to TRAP-negative MNCs. Furthermore, 1-(5-isoquinolinyl-sulfonyl)2-methylpiperazine (H-7), a protein kinase-C inhibitor, inhibited osteoclast-like MNC formation in a dose-dependent fashion. These results suggest that activation of protein kinase-C may play a role in osteoclast differentiation.


Effect of parathyroid hormone on arachidonic acid metabolism in mouse osteoblasts: permissive action of dexamethasone.

We examined the regulation of arachidonic acid (AA) metabolism in primary cultures of mouse osteoblasts under steady state and after acute stimulation by PTH. Both dexamethasone-treated and untreated cells were evaluated, as glucocorticoids are known to modulate some actions of PTH in osteoblasts and to affect arachidonic acid metabolism in cells in general. Cells were labeled with [3H]AA for 3 h, followed by a 20-h equilibrium period, then exposed to 10(-8) M PTH for different time periods ranging from 2-60 min. The results showed that although osteoblasts maintained in vitro in the absence of dexamethasone were responsive to PTH, as measured by cAMP production (50-fold increase), PTH had no effect on the distribution of AA in phospholipids and did not induce the release of free AA from phospholipid pools. After 7-day treatment of the cells with 4 x 10(-7) M dexamethasone, 15-min PTH stimulation resulted in a significant increase in free AA within the cells (mean +/- SE, +142 +/- 11%; n = 9) and a short-lived change in the distribution of AA within cellular phospholipids (phosphatidylethanolamine, -63 +/- 3%; phosphatidylinositol, +168 +/- 7%; phosphatidylserine, +296 +/- 50%; sphingomyelin, +220 +/- 20%; lysophosphatidylcholine, +634 +/- 31%; mean +/- SE; n = 3), despite the fact that no changes in PTH-induced cAMP production were observed. Because the release of free AA is an essential step in the production of eicosanoid metabolites that could act as second messengers, these findings suggest that corticosteroid treatment may activate signal transduction pathways for PTH which are latent in untreated cells, and thereby explain at least in part the profound effects of corticosteroids on osteoblast function.


The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.

The effects of the muscarinic cholinergic receptor agonist carbachol on intracellular calcium ion concentration ([Ca2+]i) and progesterone production was determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the calcium-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 13). There was a 4- to 8-fold increase in [Ca2+]i in 85% (n = 80) of the cells within 10 sec after the addition of a maximally stimulating concentration (2 mM) of carbachol. The initial [Ca2+]i spike was followed by a sustained, but lower, calcium elevation, with superimposed oscillations which returned to resting level after several minutes. Both phases of the calcium transient were inhibited by pretreating the cells with atropine (1 microM), pirenzepine (2 microM) or 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). The sustained phase of the response with its superimposed oscillations, but not the initial spike, was inhibited by pretreating the cells with the calcium channel blockers lanthanum (1 mM), cobalt (5 mM), or methoxyverapamil (50 microM), or by incubating the cells in calcium-free medium. Nifedipine (0.5-20 microM) did not affect the carbachol-induced calcium transient. 8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (50 microM) blocked the sustained-oscillatory phase of the carbachol-induced [Ca2+]i transient, but did not affect the initial spike when added before carbachol (2 mM). Despite its ability to stimulate [Ca2+]i surges, carbachol (10(-6)-10(-3) M) did not affect basal or LH-stimulated cAMP or progesterone production in 24-h cultures. These studies demonstrate that activation of chicken granulosa cell muscarinic receptors causes a rapid increase in [Ca2+]i through the release of Ca2+ from intracellular stores, followed by a sustained elevation of Ca2+ with superimposed oscillations caused by the influx of extracellular Ca2+. These results also indicate that an increase in [Ca2+]i in chicken granulosa cells does not alone stimulate steroidogenesis, since the carbachol-induced increases in [Ca2+]i were not accompanied by increased progesterone production.


Modulation of kidney function in conscious Pekin ducks by atrial natriuretic factor.

The influence of avian atrial natriuretic factor (ANF) on renal function was examined in conscious saltwater-acclimated Pekin ducks undergoing a steady state diuresis maintained by iv infused isotonic avian Krebs-Ringer solution at a rate of 1.0 ml/min. Synthetic chicken ANF (chANF) was applied iv at doses of 10, 50, and 90 ng/min.kg BW for 10 min and caused dose-dependent transient increases in urine flow, osmolal excretion, glomerular filtration rate, effective renal plasma flow, and fractional water clearance at decreased urinary osmolality. Using receptor autoradiography, binding sites for [125I]Bolton-Hunter-labeled chANF [( 125I]BH-chANF) were localized in both the reptilian-type glomeruli and the collecting duct system throughout the duck kidney. A RRA for [125I]BH-chANF, established using an enriched kidney membrane fraction, indicated that unlabeled chANF and human ANF competitively displaced [125I]BH-chANF with comparable potencies. ANF-induced modulation of renal salt and water elimination via glomerular and tubular receptor interactions is consistent with the concept that this hormone has a physiological role in avian volume homeostasis.


Stage-specific binding of inhibin and activin to subpopulations of rat germ cells.

Flow cytometry was used to separate and identify Sertoli and germ cell populations in primary rat testicular cultures derived from animals of different ages on the basis of cell size and DNA and lipid content. Multiparameter fluorescent evaluation of each cell preparation resulted in the assignment of specific staining patterns to Sertoli cells (diploid, high lipid content), spermatogonia (diploid, low lipid content), spermatocytes (large, tetraploid, high lipid content), and round spermatids (haploid, low lipid content). Each field was separately analyzed for inhibin and activin binding. Fluorescein isothiocyanate-conjugated activin bound with greatest intensity to spermatogonia, with little binding to leptotene or zygotene spermatocytes. Fluorescein isothiocyanate-conjugated inhibin bound to all stages of germ cells tested. Cross-competition data indicate that at least two and probably three distinct receptors exist for these peptides.


Luteinizing hormone-releasing hormone and neuropeptide Y influence deoxyribonucleic acid replication in three anterior pituitary cell types. Evidence for mediation by growth factors released from gonadotrophs.

Treatment of anterior pituitary reaggregate cell cultures from 14-day-old female rats with physiological doses of the gonadotropin-releasing hormone LHRH or neuropeptide Y (NPY) for 40 h dose-dependently increased [3H]thymidine ([3H]T) incorporation into DNA of cells expressing PRL immunoreactivity (PRL-ir) and of those expressing ACTH-ir, whereas these peptides decreased the number of [3H]T-labeled cells expressing GH-ir. The effects of NPY were of the same magnitude as those of LHRH. The effects of LHRH were not seen in a gonadotroph-deprived cell population obtained by sequential velocity and buoyant density gradient sedimentation. When the latter cell population was coaggregated with purified gonadotrophs from 14-day-old rats, LHRH did enhance [3H]T labeling of lactotrophs and decreased that of somatotrophs. Gonadotroph-conditioned medium obtained by continuous perifusion of gonadotroph-rich reaggregates contained four different high molecular weight substances mimicking the effects of LHRH and NPY on [3H]T incorporation in the respective pituitary cell types. These substances were partially purified and separated from each other by concentration on a Bond-elut C18-reversed phase cartridge, ultrafiltration, and C18-reversed phase HPLC. One factor stimulated [3H]T labeling of lactotrophs, another that of corticotrophs, and two others inhibited [3H]T labeling of somatotrophs. The present data suggest that the development of PRL-, GH-, and ACTH-containing cells in the pituitary is modulated by LHRH and/or NPY and that the action of LHRH and probably also of NPY is mediated by specific paracrine growth factors released from gonadotrophs.


Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes.

Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide shown to be cosecreted with insulin from the pancreatic beta-cells. We have investigated the existence and characteristics of IAPP binding sites in the rat. Specific binding sites for [125I]IAPP were found to be highest in the lung followed by the stomach fundus, spleen, brain stem, hypothalamus, and the liver, respectively. The interaction of [125I]IAPP with its binding site was rapid and temperature dependent, displaying optimum binding at 4 C. This may be explained by the rapid degradation of the label observed at 22 C and 37 C, as determined by fast protein liquid chromatography analysis, and also degradation of the receptor at 37 C. Binding of [125I]IAPP was rapidly dissociated by the addition of 200 nM unlabeled peptide. The presence of nonmetabolizable GTP-gamma-S (0.5 microM) reduced binding, thus suggesting the coupling of the binding site to a G protein. Rat IAPP displaced [125I]IAPP displaying an IC50 of 5.75 x 10(-9) M (mean, n = 4). Displacement was also seen with human IAPP (IC50 = 5.53 x 10(-8) M), human alpha-calcitonin gene-related peptide (CGRP) (IC50 = 3.8 x 10(-8) M), rat alpha-CGRP (IC50 = 9.0 x 10(-7) M), and rat beta-CGRP (IC50 = 5.53 x 10(-8) M); suggesting an IAPP-specific binding site. Scatchard plots for rat IAPP binding in the lung gave a dissociation constant of 10.4 +/- 2.63 nM (mean +/- SE, n = 4) and maximal binding of 3.1 +/- 0.97 pmol/mg (mean +/- SE, n = 4), displaying a single class of binding site. Chemical cross-linking analysis showed binding of IAPP to sites of Mr 67,000, 64,000, and 38,000. These findings suggest that specific IAPP binding sites exist which differ from the CGRP receptors in rat tissues. This indicates a possible novel autocrine/paracrine role for IAPP.


Indirect evidence to suggest that prolactin mediates the adrenal action of haloperidol to stimulate aldosterone and corticosterone secretion in rats.

The effect of dopamine-antagonists on steroid secretion has revealed conflicting results regarding the confirmation of in vivo findings in vitro. In order to discriminate in vivo systemic and local action of the dopamine-antagonist haloperidol (HAL) on aldosterone and corticosterone secretion, microdialysis of the adrenal cortex in conscious, freely moving rats was employed. The effects of 2.5 mg HAL ip or intraadrenal dialysis of 20 micrograms/ml HAL in rats with an intact pituitary gland on PRL, aldosterone, and corticosterone secretion were examined. Systemic HAL application resulted in a 40-fold increase in PRL secretion and stimulated aldosterone and corticosterone production significantly. In contrast, intraadrenal dialysis of HAL had no effect on the secretory pattern of PRL or either steroid hormone, indicating no direct drug action on cells of the rat adrenal cortex. Similarly, ip injection of 2.5 mg HAL in hypophysectomized rats did not alter PRL or steroid hormone levels. We conclude that the dopamine-antagonist HAL stimulates aldosterone and corticosterone secretion in rats through a pituitary factor, probably PRL, but not through direct effects at the adrenal cortex.


The effect of insulin-induced hypoglycemia on gene expression in the hypothalamic-pituitary-adrenal axis of the rat.

This study has examined the effects of insulin-induced hypoglycemia on expression of the CRH, arginine vasopressin, and POMC genes and corresponding peptides in freely moving, unanesthetized, male Sprague-Dawley rats. Animals were infused with 150 mM NaCl for 3 days before the experimental day and were then administered insulin (4 U/kg) or saline iv. In one experiment animals were killed 0, 30, 60, or 90 min after insulin or saline, and RNA was isolated from anterior pituitary, cerebral cortex, and punches of the hypothalamic paraventricular and supraoptic nuclei. In a second experiment, animals were killed 90 min after insulin or saline treatment, and RNA was isolated from whole hypothalami. RNA was analyzed by Northern blot. Plasma glucose fell from 106 +/- 5 to 38 +/- 2 mg/dl after insulin administration and remained low for the duration of the experiment. Plasma levels of ACTH, corticosterone, and vasopressin were 10-, 6-, and 4-fold higher, respectively, in the insulin-treated vs. control animals (by analysis of variance, P less than 0.0001 in all cases), while plasma CRH was unchanged. During hypoglycemia POMC mRNA levels were 1.8-fold higher in the insulin-treated group (by analysis of variance, P less than 0.025). In contrast, paraventricular nucleus, whole hypothalamic, and parietal cortex CRH mRNA and vasopressin mRNA were unchanged. These data support previous studies which indicated that POMC gene expression is increased by hypoglycemia. However, we found no evidence for an increase in paraventricular nucleus or cerebral cortex CRH mRNA expression during hypoglycemia-associated stimulation of the hypothalamic-pituitary-adrenal axis, suggesting that another factor(s) may mediate the observed increase in POMC gene expression.


Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition.

Biotinyl analogues of rat amylin were synthesised with sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate (NHS-SS-Biotin). Biotinylated amylin peptides were purified by HPLC, quantitated, and the presence of the biotin group at Lys-1 confirmed by peroxidase-labelled avidin and FAB mass spectroscopy. Amylin-biotin retained a similar affinity for binding to rat liver plasma membranes compared with rat amylin and also completely inhibited insulin-stimulated glycogen synthesis in rat soleus muscle incubated in vitro. These biologically active amylin probes will enable a complete analysis of amylin/CGRP receptor expression in various cell types and facilitate the isolation and characterisation of the hormone-receptor complex.


Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C.

A large Mg2+ cell uptake against concentration gradients is stimulated in collagenase-dispersed rat myocytes by carbachol and in hepatocytes by carbachol or vasopressin. The signalling pathway(s) responsible for this stimulation of Mg2+ uptake was investigated by using various activators or inhibitors of protein kinase C (PKC) and by correlating Mg2+ uptake with cell PKC activity and cAMP content. In both cell preparations, the direct stimulation of PKC by diacylglycerol analogs or phorbol esters reproduce the same pattern of Mg2+ uptake as that induced by carbachol or vasopressin. These data indicate that the activation of PKC is responsible for a stimulation of Mg2+ uptake by myocytes or hepatocytes, whereas increase in cAMP in these cells stimulates Mg2+ release.


Differential regulation of metalloprotease steady-state mRNA levels by IL-1 and FGF in rabbit articular chondrocytes.

The expression of collagenase and stromelysin is believed to be coordinately regulated. In this report however, we provide evidence that suggests subtle differences may exist in the early events of the induction of these enzymes. Rabbit articular chondrocytes treated with interleukin-1, either alone or in combination with fibroblast growth factor, accumulated steady-state mRNA levels for both the enzymes, with the latter treatment more effective in inducing greater levels and within a shorter time. Further, the induction of the enzymes by either protocol was blocked by cycloheximide co-treatment. Cycloheximide added 1 h post-stimulation with interleukin-1 + fibroblast growth factor failed to block stromelysin mRNA expression, but was able to block collagenase steady-state mRNA levels. Transforming growth factor-beta, another inhibitor of metallprotease induction, showed no such differential activity. The results suggest that collagenase and stromelysin may have subtle variations in their induction pathways. Our studies further show that the enzyme induction by interleukin-1 alone or in combination with fibroblast growth factor occurs through different, but related mechanisms.


Oxidation of cytosolic NADH by the malate-aspartate shuttle in HuH13 human hepatoma cells.

1. The reoxidation of cytosolic NADH was studied in a line of human hepatoma cells (HuH13) whose mitochondria preferentially utilized glutamine for ATP formation. 2. The tumor cells showed mitochondrial reoxidation of NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain was demonstrated by the addition of specific inhibitors. 3. Glutamine oxidation proceeded in the tumor mitochondria exclusively via a pathway involving transamination. Malate stimulated aspartate production from glutamine. 4. When the tumor cells were cultured in Eagle's medium with aminooxyacetate or in the absence of glutamine, a marked reduction in the cellular NAD/NADH ratio was observed. 5. These results indicate that the malate-aspartate shuttle was functioning in the tumor cells.


Neonatal status convulsivus, spongiform encephalopathy, and low activity of Na+/K(+)-ATPase in the brain.

The first and second child of a family died from neonatal seizures with no detectable brain malformation, metabolic, infectious, or chromosomal etiology. Neuropathological examination of the brain of the second child who died at 11 days revealed a widespread spongy state and a selective vulnerability of the astrocytes characterized by numerous enlarged bare astrocytic nuclei and different forms of astrocyte degeneration. The glial cells were strongly positive for glial fibrillary acidic protein and vimentin immunocytochemical reaction. Cortical measurement of Na+/K(+)-ATPase revealed very low enzyme activity. We hypothesize that a defect of Na+/K(+)-ATPase of the astrocytes could be the common pathogenetic factor for the congenital status convulsivus and for the spongy state.


Theoretical study about the variability of the genome of foot-and-mouth disease viruses.

1. The possibilities of change in amino acids of a protein are discussed in terms of a point mutation. 2. Whereas Met and Trp are forced to change due to a point mutation, other amino acids (Ala, Arg, Gly, Leu, Pro and Val) have a probability of 1/3 to survive in the sequence. 3. On basis of these considerations, the genome from 5 strains (CSP, C3Ind, O1K, A10 and A12) of the foot-and-mouth disease virus was studied. 4. A hypothetical genealogic tree for these strains is suggested, where CSP and C3Ind are close and also A10 and A12. O1K is closer to A10 and A12 than to CSP or C3Ind.


Giant axonal neuropathy with predominant central nervous system manifestations.

The authors describe a 25-year-old woman with giant axonal neuropathy (GAN) and severe CNS involvement. She had been admitted to hospital with generalized seizures, and had gait disturbances followed by progressive mental deterioration since childhood. Neurological examination revealed mental retardation, scanning speech, cerebellar dysfunction, pyramidal signs, mainly in the lower extremities, and peripheral sensory neuropathy. Sensory nerve conduction velocity was decreased; brain CT and MRI showed diffuse demyelination. Sural nerve biopsy revealed characteristic signs of GAN. The patient's older sister had died at the age of 23, after having had similar neurological disturbances since childhood. This case illustrates an unfamiliar presentation of GAN, characterized by mild sensory neuropathy and serve CNS involvement, including seizures.


Results of the intercalibration study of laboratories involved in assessing the environmental consequences of the Chernobyl accident.

Within the framework of the International Chernobyl Project, the IAEA's Seibersdorf Laboratories organized an intercalibration exercise among some of the laboratories which were involved in assessing the environmental contamination in the USSR due to the accident. The objective was to assess the reliability of the radioanalytical data for food and environmental samples, which were used to assess the doses. In the initial study reference materials from the stocks of the IAEA's Analytical Quality Control Services (AQCS) were re-labelled and submitted to 71 laboratories as blind samples. These natural matrix materials included samples of milk (containing 2 different levels of radioactivity), soil, air filters and clover. The concentrations of radionuclides in these samples were known from previous intercalibration exercises. The overall range in performance was broad, which is similar to what has been observed in previous international intercomparisons. The results obtained by gamma-ray spectrometry tended to be somewhat underestimated, on average. On the other hand, the laboratories showed an overall tendency to overestimate 90Sr and possibly 239Pu, which were analysed radiochemically. The intercalibration exercise is continuing with nine materials, including: soil, grass, hay and milk powder contaminated with fallout from the Chernobyl accident. These materials, which were prepared by laboratories in the USSR, are now being tested by AQCS prior to future intercomparison exercises. Work with these materials is expected to continue for several years.


Procedures for the assay of alpha-particle emitters in water samples.

In this paper the problems associated with the "in situ" concentration techniques using MnO2 and Al2O3 as adsorbents are reviewed. Sampling equipments, radiochemical separation procedures, yield tracers, sources preparation and counting methods are described. The different behaviour of Ra, U, Th, Pu and Am is discussed and considered for future speciation studies.


Measurements of actinide gut-transfer factors in humans.

Measurements have been made of the gastrointestinal absorption in humans of 239Np and 242Cm administered together in citrate media. Using five volunteers, consistent results of (2.0 +/- 0.2) x 10(-4) and (1.7 +/- 0.3) x 10(-4) were obtained for Np and Cm respectively; the quoted uncertainties are the standard errors of the means. A progress report is given of work to measure the f1 value for Pu in humans. Early work suggests an f1 value of 2 x 10(-4).


Radioanalytical studies of fallout 63Ni.

Fallout of Nickel-63 (T1/2 = 100 a) produced in small amounts at nuclear weapon tests following the neutron activation of weapon construction material was investigated by studying carpets of lichen collected during 1961 to 1988 at the Lake Rogen district in central Sweden (62.3 degrees N, 12.4 degrees E). The maximal level of 63Ni in the lichen carpet, which occurred in 1964, was about 0.6 Bq kg-1, dry weight, and decreased to 0.1 Bq kg-1 in 1988. The deposition pattern for 63Ni was similar to other fallout radionuclides such as 137Cs, 90Sr and 239 + 240Pu. The concentrations of stable Ni were relatively constant at 0.5 to 1.0 microgram g-1 throughout the years resulting in, for example, a specific activity (63Ni/stable Ni) of 0.5 Bq mg-1 in 1964 and 0.1 Bq mg-1 in 1988. The total area content of 63Ni was estimated to be 1.0 Bq m-2 and the activity ratio 63Ni/60Co was estimated to be 0.03 in 1966. The Chernobyl accident in April 1986 did not significantly increase the levels of 63Ni. For the measurement of these extremely low-levels of 63Ni, 200 g of dry material (about 1 kg fresh) were ashed and leached with aqua regia after hydroxides had been precipitated with ammonia, leaving Ni in the aqueous phase. Nickel was extracted as a dimethylglyoxime complex by chloroform and back-extracted with HCl. Finally, Ni was electroplated onto copper discs from an ammonium sulfate medium at high pH. The radiochemical yield was determined by atomic absorption spectrometry of stable Ni before and after electrodeposition.(ABSTRACT TRUNCATED AT 250 WORDS)


The measurement of technetium-99 and iodine-129 in waste water from pressurized nuclear-power reactors.

The program for measuring the dispersal presence of contaminating nuclides in waste streams produced by pressurized-water-reactor (PWR) power plants required the establishment of analytical procedures for a number of nuclides. In this report the procedure to assess 99Tc and 129I content in liquid samples using liquid-scintillation counting is presented. 99Tc and 129I are adsorbed on AG 1 X2 ion-exchange resin. 129I is then eluted with NaOCl and further purified by liquid-liquid extraction using carbon tetrachloride and an aqueous solution of sodium thiosulphate. 99Tc is eluted with hot nitric acid (6 M/60 degrees C). If necessary any 60Co present in the sample is separated by liquid-liquid extraction of the cobalt-TTA complex with iso-BuOH. The overall recovery yield is 96.5 +/- 2.5% for 99Tc and 90.1 +/- 6.2% for 129I. Finally beta-activity is measured by liquid-scintillation counting. A limit of detection of 5 Bq/L can thus be obtained for both 99Tc and 129I.


Environmental radioactivity measurement intercomparisons in the UK.

The results of two recent (1989 and 1990) nationwide radioactivity-measurement intercomparisons covering a range of pure beta- and beta/gamma-emitting radionuclides are presented. Over fifty laboratories took part in the first exercise and, in general, the results were satisfactory although most laboratories confined themselves to gamma-ray measurements only. There were some areas of concern, notably decay-scheme data, statements of uncertainty and cascade-summing effects. A follow-up meeting was held to discuss these points with the participants. As a result of this meeting, a second exercise was held, covering a different range of nuclides at lower-activity levels and seeking to discover how participants had improved their procedures in the light of the results from the first exercise. The overall results of the two exercises are reviewed in the light of i) UK radioactivity-monitoring arrangements and ii) the National Measurement Accreditation Scheme (NAMAS).


The Gamma Contamination Food Factor.

We suggest that radioactive food contamination, as determined solely by a quantitative gamma-ray spectroscopic measurement, may, apart from the total activity per unit mass, be for quick reference conveniently characterized by another single figure which we call the "Gamma Contamination Food Factor" (GCFF). This factor may be defined as the ratio of the total specific activity of gamma-ray-emitting radionuclides in the food sample (except that of 40K) to the specific activity of 40K either in the sample itself or in an "average man". We discuss briefly the meaning and advantages of these definitions.


Poly(A) removal is the kinase-regulated step in tumor necrosis factor mRNA decay.

Tumor necrosis factor (TNF) is a pleiotropic biomodulator and an important inducer of certain pathophysiologic immune reactions such as granuloma formation, cachexia, and septic shock. The production of TNF by astrocytes, which may figure prominently in the development of immune responses within the central nervous system, is subject to post-transcriptional regulation. We have previously shown that in virus-stimulated astrocytes, inhibition of protein kinase C results in a specific, 10-fold decrease in TNF mRNA half-life. Here we show that the decay of TNF messages induced in the macrophage-like cell line RAW 264.7 by either virus or lipopolysaccharide was subject to similar regulation, and that this pathway influenced the amount of TNF protein released by stimulated cells. Using a modified RNase protection assay, we demonstrate that inhibition of protein kinase C significantly enhanced the rate of poly(A) removal from TNF mRNA, thus facilitating an early event in the process of mRNA degradation.


cAMP-dependent kinases in the algal flagellate Euglena gracilis.

Euglena cells grown in diurnal light-dark cycles exhibit circadian variations of their cAMP content, which we believe to be under the control of an endogenous timer because they persist in constant darkness in the absence of any environmental time cue. We think that these cAMP oscillations may play a role in the regulation of some of the numerous cellular activities that are known to display circadian rhythmicities in this organism. The role of cAMP in algal cells is still controversial, however, since the nature of the cAMP "receptor" is unknown. We show that extracts of the achlorophyllous ZC mutant of Euglena gracilis contain two cAMP-binding proteins, which bind cAMP with a high affinity (Kd values of 10 nM and 30 nM) and which can be separated by DEAE-cellulose chromatography. Protein kinase activity was assayed using Kemptide as a substrate. Stimulation of kinase activity by cAMP was observed after partial purification by DEAE-cellulose chromatography. Two peaks of activity were resolved, corresponding to distinct enzymes with different cAMP-analog specificities. Thus, cAMP signaling in plant cells may proceed by the phosphorylation of target proteins by cAMP-dependent kinases, in a manner similar to that of animal cells.


The determination of dissolved radon in water supplies by the E-PERM system (electret ionization chamber).

Elevated levels of radon may be found in well waters and individuals who ingest such waters may receive significant radiation doses. This paper discusses the experience of the Radiation Protection Service in using the E-PERM system for measuring dissolved radon in water samples.


Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.


A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites.

The first fluorescently labeled phenylalkylamine, DMBODIPY-PAA (5-(3-[3-(4,4-difluoro-5,7-dimethyl-3a, 4a-diaza-4-bora-indacen-3-yl)propionamido] phenethyl-N-methylamino)-2-isopropyl-2-(3,4,5-trimethoxyphenyl)-valer onitrile) has been introduced for L-type Ca2+ channel research. DMBODIPY-PAA binds reversibly to L-type Ca2+ channels purified from rabbit skeletal muscle microsomes by wheat germ agglutinin-Sepharose chromatography. In this preparation DMBODIPY-PAA labels 412 pmol of phenylalkylamine receptors/mg of protein with a Kd of 6.82 nM and a favorable signal-to-noise ratio. Therefore DMBODIPY-PAA has a higher affinity for purified Ca2+ channels than the commonly employed radioligands and consequently has assisted in channel purification after prelabeling by simply monitoring receptor-bound fluorescence. (+)-PN200-110 (which is stimulatory for (-)-[3H]desmethoxyverapamil binding to purified Ca2+ channels) inhibits DMBODIPY-PAA labeling. Since these drug interactions are reciprocal, the phenylalkylamine and dihydropyridine binding sites of the alpha 1-subunit are tightly coupled. Kinetic and equilibrium binding studies with (-)-[3H]desmethoxyverapamil and DMBODIPY-PAA show that phenylalkylamine binding to L-type Ca2+ channels is dependent on Ca2+. Chelation of divalent metal ions converts phenylalkylamine receptors into a very low affinity state. This conversion is temperature- and time-dependent and completely reversible (K0.5 for free Ca2+ = 58 nM). This study demonstrates the utility of fluorescent ligands for binding studies with L-type Ca2+ channels and provides evidence for coupling between Ca2+ binding sites and phenylalkylamine receptors.


Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium.

The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.


Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.

Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.


Dual mode of inhibition of purified DNA ligase I from human cells by 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.


Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.