Different forms of alkaline phosphatase in adult rat femur. Effect of a vitamin D3-deficient diet and of a sorbitol-enriched diet. In the femoral extremities of the adult rat containing the metaphysis, the epiphyseal cartilage, and the epiphysis, four alkaline phosphatase (AP) forms were distinguished on polyacrylamide gel electrophoresis. Two soluble forms were present in the 160,000 g supernatant: one of Mr 165 kDa and another of Mr 110-115 kDa, which exhibited a strong catalytical activity. Moreover, from the pellet, three membrane-bound forms of Mr 130, 110-115, and 100 kDa could be extacted with sodium deoxycholate. When denaturated AP was visualized by postelectrophoretic autoradiography of the phosphorylated intermediates, subunits always appeared as three monomers of Mr 75-80, 60-70, and 50-60 kDa. As four native forms but only three types of subunits were found to be present in the femur, it seems that, apart from homodimers, some heterodimers could also occur. Three types of diets were administered to three groups of rats for 5 weeks. Two are known to disturb bone mineralization: (1) a vitamin D3-deficient diet, and (2) the same as (1) but enriched with 12% sorbitol. The third was a normal diet containing vitamin D3. Concerning the effects on AP of dietary sorbitol and the vitamin D3-deficient diet, it was found that rats receiving the diet supplemented with sorbitol showed a substantial rise in the activity of the Mr 165 kDa form with the concomitant appearance of a new monomer of Mr 100 kDa. In contrast, rats fed the vitamin D3-deficient diet always displayed an increase in enzyme activity, principally of the Mr 100 and 110 kDa forms.(ABSTRACT TRUNCATED AT 250 WORDS)


[Pharmacological intervention in experimental multiple organ failure in rats]. This paper presents a primary study of the treatment of experimental multiple system organ failure (MSOF) in rats. The rats were pretreated with xanthine oxidase inhibitor allopurinol, the energy metabolism regulator fructose-1,6-diphosphate (FDP) and purified Chinese herbal medicine polydatin. The incidence of MSOF decreased from 71.4% in the untreated group to 35.7%, 47.1% and 16.7% in treated groups, respectively, while the mean survival time was prolonged to 38.5h, 30.2h and 41.7h in treated groups, respectively, as compared with 26.4h in the untreated group. In addition to the known antioxidant effect of the allopurinol, this study also suggests that FDP and polydatin enhance the capacity of antioxidtion.


Effects of alprazolam on the development of MTV-induced mammary tumors in female mice under stress. Female C3H/He mice carrying the mammary tumor virus (MTV) were monitored for mammary tumor incidence and latent periods while subjected to a daily intraperitoneal (i.p.) injection of placebo or alprazolam (1 mg/kg per day). Although all of the mice were potential candidates for MTV-induced breast cancer, those injected with alprazolam were partially protected against adverse effects of stress induced by the daily administration of placebo.


Crocidolite asbestos suppresses the differentiation of HL-60 cells induced by DMSO. This study was undertaken to determine if the biological function of inducers for cell differentiation is affected by asbestos fibers, which are sometimes deposited in human tissues. Protein kinase C activity, c-myc protein expression and cell surface CR3 expression were used as the markers of cell differentiation. The function of dimethylsulfoxide (DMSO), an inducer of cell differentiation, was suppressed by the co-culturing of crocidolite asbestos, because DMSO reacted with the hydroxyl radical released after the stimulation with crocidolite and spent itself. Superoxide dismutase (SOD) inhibited the effect of crocidolite, reacting rapidly with .O2- before the secondary release of .OH. Asbestos fibers deposited in tissues may inhibit the function of inducers which stimulate immature cells to differentiate, because such inducers frequently are also radical scavengers.


Cytotoxic analog of somatostatin containing methotrexate inhibits growth of MIA PaCa-2 human pancreatic cancer xenografts in nude mice. Nude mice bearing xenografts of MIA PaCa-2 human pancreatic cancer cell line were treated for 4 weeks with AN-51, a somatostatin octapeptide analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121) containing methotrexate attached to the alpha-amino group of D-Phe in position 1. Control groups of mice received saline, RC-121 or methotrexate. Drugs were given in equimolar doses by daily s.c. injections. After 7 days of treatment with 25 micrograms/day of AN-51, tumor growth was completely inhibited although the treatment had to be suspended because of toxic side effects, especially on the gastrointestinal tract, accompanied by major weight loss of the animals. Mice were allowed to recover for 1 week and treatment was continued with 12.5 micrograms/day AN-51. After 2 weeks of additional therapy, tumor volume, percentage change in tumor volume, and tumor weights were significantly decreased, compared with controls, only in the group treated with AN-51. Methotrexate and RC-121 also inhibited tumor growth, but their effects were not statistically significant. AN-51 retained its hormonal activity and decreased serum growth hormone levels in mice. Binding affinity of AN-51 for somatostatin receptors on MIA PaCa-2 cells was found to be 2.5-times lower than that of parent compound RC-121. This is the first report on inhibition of human pancreatic cancer growth in vivo by somatostatin analogs carrying cytotoxic radicals.


Early activation of the proto-oncogene c-fgr during Epstein-Barr virus immortalization. In an attempt to clarify the chronological relationships between Epstein-Barr virus (EBV) infection, B cell immortalization and c-fgr activation, we evaluated for the presence of EBV-determined nuclear antigen (EBNA), cellular DNA synthesis and expression of c-fgr-specific RNA following infection of human peripheral blood lymphocytes with B95-8 EBV. High expression of c-fgr was observed prior to EBNA detection and cellular DNA synthesis in EBV-infected cells. These results suggest that activation of c-fgr is an essential event during the early phase of EBV immortalization.


Vanadate enhances transformation of bovine papillomavirus DNA-transfected C3H/10T1/2 cells. Bovine papillomavirus (BPV) DNA-transfected C3H/10T1/2 cells respond to tumor promoters by enhanced production of transformed foci. Vanadate, a suspected carcinogen, is a mitogen, generates active oxygen species and alters phosphorylation of proteins. We investigated whether vanadate would enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Transformed foci in BPV DNA transfected C3H/10T1/2 cells exposed continuously to vanadate for 21 days increased in a dose-dependent manner to 50-fold at 4 microM vanadate. This increase was not due to enhanced uptake of BPV DNA post transfection. Neither catalase nor superoxide dismutase inhibited the vanadate-mediated increase in transformed foci but this does not necessarily rule out the involvement of intracellular active oxygen species. At vanadate concentrations greater than 6 microM, cells lost adherence to the Petri plates. We conclude that vanadate is capable of enhancing BPV DNA-mediated cell transformation. Possible mechanisms may involve active oxygen species or altered patterns of protein phosphorylation.


Induction of retinoic acid-binding protein in normal and malignant human myeloid cells by retinoic acid in acute promyelocytic leukemia patients. Retinoic acid has striking effects on development and cell differentiation. Its biological effect is a highly regulated process that is controlled by specific proteins. In the nucleus, different retinoic acid receptors have been identified and their genes cloned. In the cytosol, retinoid binding proteins, cellular retinoic acid-binding protein and cellular retinol-binding protein, have been correlated with normal and malignant tissue differentiation. Recently, differentiation therapy of acute promyelocytic leukemias (AML3 subtype) with all-trans-retinoic acid has been shown to be an efficient alternative to chemotherapy. The retinoic acid receptor alpha gene has been shown to be specifically rearranged in AML3 through the t(15;17) translocation. The molecular basis of the effect to reverse the leukemic phenotype of all-trans-retinoic acid is not yet elucidated. To further study retinoic acid efficacy in AML3 leukemia, retinoic acid-binding proteins were studied in the cytosol extracts of hematopoietic cells. No retinoic acid binding activity was detected in normal or malignant hematopoietic cells whether sensitive or not to retinoic acid. However, detectable binding to a cytosolic protein corresponding to cellular retinoic acid-binding protein (M(r) 15,000, Kd 3 nM) was observed in the bone marrow cells of AML3 patients undergoing all-trans-retinoic acid therapy. We suggest that both the induction and subsequent presence of cellular retinoic acid-binding protein may influence the therapeutic efficacy of retinoic acid and must be taken into account when studying its effect in acute promyelocytic patients.


K-sam-related gene, N-sam, encodes fibroblast growth factor receptor and is expressed in T-lymphocytic tumors. We recently reported the isolation of the K-sam complementary DNA (cDNA), which was amplified preferentially in poorly differentiated types of stomach cancer and codes for one of the heparin-binding growth factor or fibroblast growth factor (FGF) receptor families. The K-sam-related gene, N-sam (NCC-IT-cell-derived sam), was isolated by screening of the cDNA libraries of human immature teratoma cells, NCC-IT. Sequence analysis of the N-sam cDNAs showed that N-sam encodes a human FGF receptor, the FLG protein. N-sam was expressed in lymphocytic leukemia/lymphoma cells, predominantly in the thymic T-cell phenotype. In a T-cell leukemia line, MOLT3, N-sam mRNA expression was markedly enhanced by 12-O-tetradecanoylphorbol-13-acetate treatment and was also up-regulated by basic FGF exposure. These results indicate that N-sam expression is regulated during T-cell ontogeny and modulated by its putative ligand exposure. The results also suggested that interaction between immature T-cell and marrow or thymic interstitial cells might be mediated by N-sam and basic FGF stored in the extracellular matrix of stromal cells.


Loss of colonic HLA antigens in familial adenomatous polyposis. The loss of HLA antigens by neoplastic cells is considered important for tumor growth and metastasis, since it may allow tumors to escape immune surveillance. We studied the expression of HLA class I and II antigens in the colons of 10 patients with familial adenomatous polyposis (FAP), a condition which leads inevitably to colorectal cancer. Expression of HLA class antigens was studied by immunohistochemistry in (a) adenomas from patients with FAP, (b) histologically normal mucosa distant from the adenomas, and (c) histologically normal colonic mucosa from normal subjects. The expression of HLA class I and II antigens was decreased in histologically normal mucosa from FAP patients compared to normal controls. Adenomas showed a similar but quantitatively more pronounced reduction (or loss) of HLA antigen expression. The reduction of HLA expression in adenomas was comparable to that observed in sporadic colon carcinomas. This generalized suppression of HLA gene expression in the colon of FAP patients, which precedes the onset of overt histological manifestations of neoplasia, may be an important early event in colon carcinogenesis.


Expression of c-sis/platelet-derived growth factor B, insulin-like growth factor I, and transforming growth factor alpha messenger RNAs and their respective receptor messenger RNAs in primary human gastric carcinomas: in vivo studies with in situ hybridization and immunocytochemistry. In situ hybridization and immunocytochemistry have been applied to investigate the expression of c-sis/platelet-derived growth factor (PDGF)-B, insulin-like growth factor (IGF)-I, and transforming growth factor alpha mRNAs and their respective receptor mRNAs in three primary human gastric carcinomas and in their adjacent nonmalignant mucosas. Expression of c-sis/PDGF-B mRNA and PDGF-receptor beta mRNA was seen in the tumor cells of the three gastric cancer specimens but not in their adjacent nonmalignant mucosa. The mRNA expression was accompanied by the expression of their respective protein products. IGF-I, IGF-I receptor, and epidermal growth factor receptor mRNAs were seen in both the tumor cells of the gastric cancer specimens and in nonmalignant mucosa. Transforming growth factor alpha mRNA was expressed in gastric tumor cells but not in nonmalignant mucosa. The coexpression of a potent "competence" growth factor, PDGF, and "progression" growth factors, IGF-I and transforming growth factor alpha, in the tumor cells of gastric carcinomas may contribute to their growth and maintenance.


[Malignant transformation in multiple eccrine spiradenoma]. Development of a poorly differentiated eccrine carcinoma was observed in a 6-year-old woman. She had been operated on many times during 20 years for some tens of classical as well as less usual forms of eccrine spiradenomas, e.g. giant vascular spiradenomas. They were mostly localized in the skin of back, thorax and neck. The patient died of an extensive skin involvement and spine and liver secondaries 12 months after occurrence of the carcinoma. The structure of carcinoma was trabecular and partly papillary, tumour cells had bulky nuclei and striking nucleoli. There was a juxtaposition of spiradenomas with carcinoma and direct transformation of spiradenoma into carcinoma was observed. Immunohistological positivity of carcinoma concerned S-100 protein, slightly CEA, focally cytokeratin 7 and 18; cytokeratin 14 was negative. Ultrastructure of tumour cells showed irregular intercellular lumina with some microvilli, but structures characteristical for eccrine glandular of ductal differentiation were lacking.


Enhancement of rat hepatic macrophages by treatment with interleukin-2 and streptococcal preparation OK432, with reference to antitumor activity, soluble factor production and Ia expression. The effect of biological response modifiers, such as interleukin-2 (IL-2) and streptococcal preparation OK432, on the functions of hepatic macrophages was investigated. The macrophages, even with no exogenous stimulation, produced superoxide anion (O2-) and tumor necrosis factor (TNF), displayed cytotoxicity against K562 cells and cytostasis against P815 cells and expressed immune-region-associated antigen (Ia). IL-2 administered in vitro or in vivo enhanced O2- production by hepatic macrophages and the intravenous injection of OK432 also enhanced O2 production. Furthermore, IL-2 added to the culture medium of hepatic macrophages isolated from OK432-injected rats augmented O2- production even more. The TNF production and Ia expression of the macrophages were also increased by the intravenous injection of OK432. As with O2- production, the cytotoxicity of the cells was enhanced by OK432 injection or by IL-2 added to the culture medium and the combination of OK432 and IL-2 augmented their cytotoxicity even more. Thus, the present study suggested that IL-2 and OK432 induce the augmentation of the antitumor activity of hepatic macrophages, partly as a result of the increase in production of O2- and TNF and Ia expression.


Proton efflux from rat skeletal muscle in vivo: changes in hypertension. 1. An analysis of the recovery kinetics of intracellular pH and phosphocreatine concentration after exercise in skeletal muscle was developed to calculate the rate of proton efflux in vivo. 2. Recovery of rat leg muscle pH after sciatic nerve stimulation was faster in spontaneously hypertensive rats than in Wistar-Kyoto controls (both n = 5). 3. Analysis of these data showed that the rate of proton efflux depends on intracellular pH, being greater at lower pH. 4. The early rate of proton efflux was greater in spontaneously hypertensive rats [measured over the first 0.8 min, 12.5 mmol min-1 kg-1 (SEM 1.8) in spontaneously hypertensive rats compared with 7.6 mmol min-1 kg-1 (SEM 0.4) in Wistar-Kyoto rats, P less than 0.05], even though pH at the start of recovery was higher [6.30 (SEM 0.03) in spontaneously hypertensive rats compared with 6.17 (SEM 0.01) in Wistar-Kyoto rats, P less than 0.01]. 5. This novel analysis provides a quantitative estimate of the rate of proton efflux in vivo, and demonstrates directly that this is increased in spontaneously hypertensive rats, as has previously been inferred from pH changes during exercise and studies of cultured muscle cells in vitro.


Platelet cytosolic free calcium concentration, total plasma calcium concentration and blood pressure in human twins: a genetic analysis. 1. We used path analysis and maximum-likelihood model fitting to evaluate the relative contributions of genetic and environmental factors to the relationships observed between level of blood pressure and both total plasma calcium concentration and platelet cytosolic free calcium concentration in 109 twin pairs. 2. Total plasma calcium concentration was positively associated with systolic (r = 0.26, P less than 0.001) but not diastolic blood pressure, a relationship which remained significant after adjustment for albumin, age and body mass index. A relationship between platelet cytosolic free calcium concentration and both systolic and diastolic blood pressure (r = 0.17 and r = 0.13, respectively, P less than or equal to 0.05) was no longer significant after adjustment for age and body mass index. 3. Additive genetic influences, unique environmental effects and age contributed to 60%, 30% and 10% of the variance in systolic blood pressure, respectively. Additive genetic effects explained 78% of the variance in plasma total calcium concentration and at least 48% of the variance in platelet cytosolic free calcium concentration in females and 37% in males. 4. Bivariate factor models provided evidence of genetic, but not environmental, co-variation of total plasma calcium concentration and systolic blood pressure, suggesting that a common genetic factor (or factors) contributes to their univariate relationship. In contrast, there was evidence of environmental, but not genetic, covariation of platelet cytosolic free calcium concentration and systolic blood pressure, suggesting that some of the individual experiences specific to each twin may be causing these two traits to vary together. 5. The possible confounding effects of adiposity and environmental factors should be considered in future studies investigating the role of intracellular calcium levels in the pathogenesis of hypertension.


Effects of oral contraceptives containing oestrogen combined with norethisterone or levonorgestrel on erythrocyte cation transport in normal women. 1. Studies were undertaken in pre-menopausal women to examine the effects of treatment with standard oestrogen-progestogen and progestogen-ony oral contraceptives on erythrocyte Na+,K+ co-transport and Na(+)-Na+ countertransport over 3- and 6-month periods. Concurrent observations were made on other erythrocyte cation transport components, plasma lipid concentrations, plasma renin activity, plasma aldosterone concentration and blood pressure. 2. Na+,K+ co-transport, measured as the ouabain-resistant, frusemide-sensitive component of 86Rb+ influx, and Na(+)-Na+ countertransport, measured as the ouabain-resistant, phloretin-sensitive component of 22Na+ influx, were both increased in women taking, on days 1-21 of their cycle, ethinyloestradiol (30-50 micrograms) combined with norethisterone (1000 micrograms or 500-1000 micrograms) for 3 or 6 months. Neither of these fluxes was increased in a control group of women, or in women treated for the same time periods with ethinyloestradiol combined with levonorgestrel. 3. In a separate study of erythrocyte cation transport (excluding Na(+)-Na+ countertransport), in which women undertook treatment with norethisterone only (350 micrograms/day) for 6 months starting 6 weeks post partum, no changes in Na+,K+ co-transport were observed at 3 or 6 months; there were no changes in cation transport in a corresponding control group. 4. The results of these studies confirm that certain oral contraceptive compounds can alter erythrocyte cation transport, and indicate that norethisterone in higher dose preparations is the component predominantly responsible. The alterations observed could not be explained by a direct link with concurrent changes in plasma triacylglycerol concentrations or in the renin-aldosterone axis and were not closely associated with elevation of blood pressure.


Central bradykininergic system in normotensive and hypertensive rats. 1. The kinin antagonist des-Arg9-[Leu8]bradykinin, injected into the lateral ventricle, caused a long-lasting, dose-dependent reduction in arterial blood pressure and heart rate in spontaneously hypertensive rats but not in normotensive Wistar-Kyoto rats; the antagonist also blocked the pressor response to ventricularly infused bradykinin in both strains. 2. Bradykinin content was increased in the hypothalamus and septum and decreased in the dorsal medulla of spontaneously hypertensive rats when compared with those of normotensive Wistar-Kyoto rats, whereas similar bradykinin contents were observed in the pineal gland, hypophysis and rostroventrolateral medulla of both rat strains. 3. Increased concentrations of bradykinin and its precursor kininogen were found in the cerebrospinal fluid of spontaneously hypertensive rats. 4. Bradykinin receptor numbers, measured as the binding of [125I-Tyr1]bradykinin to nervous tissue, were found to be increased in the dorsal medulla and hypophysis, and to be decreased in the pineal gland, of spontaneously hypertensive rats. 5. Therefore, the central kinin system may participate, by both pre- and post-synaptic mechanisms, in the maintenance of hypertension in spontaneously hypertensive rats.


Cutaneous vascular responses and thermoregulation in relation to age. 1. Sympathetic vasoconstrictor responses to inspiratory gasp and contralateral arm cold challenge were assessed in fingertip skin in relation to age and were correlated with vasoconstrictor ability during body cooling. The above relationship was also examined in diabetic patients in whom vasoconstrictor responses to inspiratory gasp and contralateral arm cold challenge had been shown previously to be markedly impaired. 2. Vasoconstrictor responses to inspiratory gasp and contralateral arm cold challenge, measured by laser Doppler flowmetry, were significantly reduced in the elderly group, although individual responses varied from normal to absent, and they also had a considerably greater variability as measured on three separate occasions than seen in young subjects. Discriminant analysis showed that, from each of three occasions, 65% of vasoconstrictor responses were abnormal in the elderly group. 3. Body cooling was performed by reducing the environmental temperature from 40 degrees C to 12 degrees C, and the time taken for blood flow to fall to 75%, 50% and 25% of the pre-cooling level (VC75, VC50, VC25, respectively) was calculated. Vasoconstriction was rapid in young subjects and was consistent with good vasoconstrictor responses to inspiratory gasp and contralateral arm cold challenge. In the elderly group, vasoconstriction was slower, but only the VC25 value differed significantly [elderly group, 13.3 (7.9-31.0) min, young group, 5.7 (2.7-15.5) min; median (interquartile range); P less than 0.05].(ABSTRACT TRUNCATED AT 250 WORDS)


An objective test for the diagnosis and grading of vasospasm in patients with Raynaud's syndrome. 1. Reliable objective tests for the diagnosis and grading of vasospasm would be helpful in the assessment of patients with Raynaud's syndrome. 2. Measurements of finger blood flow at local finger temperatures from 32 degrees C down to 20 degrees C did not reliably distinguish between patients with Raynaud's syndrome and matched control subjects. 3. Using laser Doppler flowmetry to detect blood cell flux in fingertip skin, there was no significant difference (Wilcoxon's signed rank test) in the finger systolic blood pressure of 28 patients with Raynaud's syndrome and their matched controls when the fingers were warm at 32 degrees C. 4. Absence of flux was considered to indicate complete vasospasm and the degree of cooling required to abolish flux indicated the severity of the vasospastic condition in an individual patient. 5. Finger cooling for 5 min did not significantly alter finger systolic blood pressure in the control subjects, but abolished blood cell flux in the fingertip skin of 27 of the 28 patients with Raynaud's syndrome. 6. A grading scale was derived from the flux measurements. There was a significant correlation (r = 0.75, P less than 0.001) between the grading of disease severity as judged by the flux test and the clinical grade as assessed before the laboratory visit. 7. There was one false-negative result in the 28 patients with Raynaud's syndrome tested and no false-positive results in 28 matched control subjects. 8. This type of testing may prove helpful in the diagnosis and grading of vasospastic disorders.


Plasma platelet-activating factor degradation in patients with severe coronary artery disease. 1. Platelet-activating factor is inactivated in plasma by the action of a specific acetylhydrolase that cleaves the acetate moiety from the sn-2 position. Degradation was determined under optimized conditions and under conditions closer to those which may occur in vivo. The latter, or platelet-activating factor half-life, was measured by a modified method that is simple, inexpensive and reliable. 2. A hyperbolic relationship was found to exist between the two measures of degradation, the values in both normal subjects and patients with coronary artery disease falling on the tail of the hyperbola. Thus, there is an increase in platelet-activating factor half-life associated with a lowering of acetylhydrolase activity, but this increase is relatively small. 3. There were significant direct linear relationships between acetylhydrolase activity and serum total cholesterol and low-density-lipoprotein-cholesterol concentrations in both subject groups. Although acetylhydrolase activity was most closely associated with the low-density-lipoprotein-cholesterol fraction, the activity for a given serum level of low-density-lipoprotein-cholesterol was higher in patients with coronary artery disease.


Lithium pretreatment affects renal and systemic responses to angiotensin II infusion in normal man. 1. Renal and systemic responses to infusion of angiotensin II (1.25 and 2.5 ng min-1 kg-1 body weight) were examined in ten normal males 12 h after single doses of 750 mg of lithium carbonate, 250 mg of lithium carbonate (n = 6) or placebo. 2. Baseline mean arterial pressure [mean (SEM)] was higher after 750 mg of lithium [93.1 (1.7) versus 89.5 (1.9 mmHg, P = 0.014], and the subsequent rise in blood pressure during angiotensin II infusion was lower [8.2 (1.8) versus 12.2 (2.4) mmHg, P less than 0.02]. 3. Lithium at a dose of 750 mg increased overnight urinary sodium excretion before the study. The fall in fractional sodium excretion during angiotensin II infusion was reduced after pretreatment with 750 mg of lithium [750 mg of lithium, 2.73 (0.24) to 1.34 (0.08)%; placebo, 2.69 (0.26) to 1.01 (0.11)%; P = 0.02]. The increases in effective filtration fraction [750 mg of lithium, 5.4 (1.0)%; placebo, 8.6 (0.7)%; P less than 0.05] and total effective renal vascular resistance [750 mg of lithium, 3700 (390) dyn s cm-5; placebo 5100 (460) dyn s cm-5; P = 0.03] during angiotensin II infusion were also attenuated after 750 mg of lithium. Responses after 250 mg of lithium did not differ from those after placebo. 4. The fall in plasma renin activity and the increase in plasma aldosterone concentration during angiotensin II infusion were similar on each study day. 5. Renal responses to exogenous angiotensin II are altered after pretreatment with a 750 mg dose of lithium in normal man. This dose of lithium is not an inert marker of sodium handling.


Altered renal response to enhanced endogenous 5-hydroxytryptamine after tryptophan administration in essential hypertension. 1. To examine the pathophysiological significance of 5-hydroxytryptamine (serotonin) in essential hypertension, we compared the renal response to intrarenally formed 5-hydroxytryptamine by oral dosing with its precursor, L-tryptophan (2 g), in nine patients with essential hypertension and in six subjects with normotension. 2. Before tryptophan administration, urinary excretion of 5-hydroxytryptamine was significantly higher in the hypertensive group than in the normotensive group (66 +/- 8 versus 36 +/- 6 ng/min, P less than 0.05), whereas renal plasma flow and glomerular filtration rate did not differ between the two groups. After dosing with tryptophan, urinary excretion of 5-hydroxytryptamine significantly increased to the same plateau level in both groups (366 +/- 55 ng/min in the hypertensive group and 365 +/- 64 ng/min in the normotensive group). Significant and equivalent decreases in renal plasma flow were observed in the early phase after tryptophan administration in both groups (-8.5 +/- 3.4% in the hypertensive group and -8.2 +/- 1.7% in the normotensive group). Thereafter, renal plasma flow increased to above the baseline value in normotensive subjects, whereas this late vasodilatation was absent in the hypertensive group. Glomerular filtration rate significantly decreased at the time of the fall in renal plasma flow in the normotensive group (106.8 +/- 7.8 to 92.7 +/- 8.5 ml min-1 1.73 m-2, P less than 0.05), whereas it remained unchanged in the hypertensive group (108.2 +/- 6.2 to 110.4 +/- 6.3 ml min-1 1.73 m-2, not significant).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of bicarbonate administration on skeletal muscle intracellular pH in the rat: implications for acute administration of bicarbonate in man. 1. The effect of bicarbonate administration on the intracellular pH of rat skeletal muscle was examined by using 31P n.m.r. 2. Bicarbonate administered intraperitoneally caused a significant intracellular acidosis in rat skeletal muscle in vivo. When the bicarbonate was administered intravenously there was no such change in the pH of the skeletal muscle. 3. Bicarbonate administration by either route resulted in an elevated mixed venous partial pressure of carbon dioxide and an elevated arterial pH, but no significant change in the arterial partial pressure of carbon dioxide. The increase in arterial bicarbonate concentration after intraperitoneal injection of bicarbonate was delayed when compared with that after intravenous injection. 4. The administration of hypertonic solutions intravenously caused a transient 40-50% fall in blood pressure, which had resolved within 1 min. 5. The data suggest that the effect of bicarbonate administration on intracellular pH in vivo is related not only to carbon dioxide loading of the cell but also to the rate of change in the extracellular bicarbonate concentration.


Acute metabolic and hormonal responses to the inhibition of lipolysis in non-obese patients with non-insulin-dependent (type 2) diabetes mellitus: effects of acipimox. 1. Increased rates of fatty acid oxidation are frequently observed in patients with non-insulin-dependent diabetes mellitus and may contribute to hyperglycaemia by both decreasing peripheral glucose disposal and, more importantly, by increasing the rate of gluconeogenesis and therefore hepatic glucose output. Despite this relationship between lipid and carbohydrate metabolism, fasting glucose concentrations do not fall acutely in patients with non-insulin-dependent diabetes mellitus when plasma non-esterified fatty acid concentrations and lipid oxidation rates are decreased, questioning the importance of this interaction to glycaemic control. We have therefore measured the acute changes that occur 120-150 min after administration of 500 mg of the antilipolytic agent acipimox in eight non-obese male patients with non-insulin-dependent diabetes mellitus. 2. After administration of acipimox, lipolysis was inhibited as reflected by lower plasma non-esterified fatty acid (0.05 +/- 0.02 versus 0.55 +/- 0.05 mmol/l, P less than 0.001) and blood glycerol (8 +/- 1 versus 56 +/- 8 mumol/l, P less than 0.001) concentrations. The lipid oxidation rate was decreased (0.63 +/- 0.05 versus 1.02 +/- 0.08 mg min-1 kg-1, P less than 0.001), whereas there was a significant increase in the carbohydrate oxidation rate (1.93 +/- 0.17 versus 1.22 +/- 0.18 mg min-1 kg-1, P = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of epidermal growth factor and glutamine-supplemented parenteral nutrition on the small bowel of septic rats. 1. The effects of parenteral nutrition with or without glutamine supplementation and epidermal growth factor treatment (0.15 microgram/g body weight) was studied in the small bowel of septic rats after 4 days. 2. Septic rats infused with glutamine-supplemented parenteral nutrition with or without epidermal growth factor treatment survived sepsis significantly better than other septic rats given parenteral nutrition. The cumulative percentage of deaths over 4 days in septic rats infused with glutamine-supplemented parenteral nutrition was 20% (without epidermal growth factor) and 15% (with epidermal growth factor) compared with 50% in septic rats treated with parenteral nutrition without glutamine and 35% in septic rats given parenteral nutrition without glutamine but with epidermal growth factor treatment. 3. Glutamine-supplemented parenteral nutrition with or without epidermal growth factor treatment resulted in improved nitrogen balance in septic rats. The cumulative nitrogen balance over the 4 day period was the least negative as compared with other groups of septic rats. 4. Septic rats given parenteral nutrition with glutamine, epidermal growth factor or glutamine and epidermal growth factor exhibited marked increases in intestinal net rates of utilization of glutamine (P less than 0.001) and production of ammonia (P less than 0.001) compared with septic rats given parenteral nutrition without glutamine and/or epidermal growth factor treatment. 5. Septic rats given parenteral nutrition with glutamine, epidermal growth factor or glutamine and epidermal growth factor exhibited significant increases in jejunal wet weight (by 32.4-40.6%), DNA content (by 24.2-34.7%), protein content (by 29.1-50.0%), villus height (by 16.3-26.4%) and crypt depth (by 20.3-29.6%) compared with other groups of septic rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Growth regulatory peptides in gastric mucosa. 1. Epidermal growth factor and the related peptide transforming growth factor alpha have been implicated in the stimulation of gastric mucosal proliferation. We assessed the immunohistochemical distribution of these peptides and their receptor, epidermal growth factor receptor, in mucosa from the antrum and body of the stomach from 28 patients. Twenty-three of the 56 biopsies were histologically normal (12 antrum and 11 body), whereas the other 33 showed varying degrees of inflammation. 2. Epidermal growth factor, transforming growth factor alpha and epidermal growth factor receptor had maximal density of distribution on the apical surfaces of the superficial epithelial cells, but were also expressed to a lesser extent on neck and body cells of the glandular tissue (P less than 0.05). We also demonstrated that epidermal growth factor expression was greater in the epithelial cells of inflamed mucosa than in those of normal mucosa (P less than 0.05). 3. We assessed mucosal proliferation by the Ki-67 labelling index. Ki-67-positive cells were found predominantly in the neck area of the glands and were more frequent in glandular antral tissue than in body glandular tissue (P less than 0.05). Expression of epidermal growth factor receptor in the neck and isthmus cells had a significant correlation with the Ki-67 labelling index (P less than 0.05). 4. We conclude that epidermal growth factor and epidermal growth factor receptor may be important in the adaptation of gastric mucosa to inflammation.


Absorption of human calcitonin across the rat colon in vivo. 1. We studied the absorption of human calcitonin across the colon of juvenile female rats in vivo. Both pharmacokinetic and pharmacodynamic parameters were monitored to measure absorption. 2. Intracolonically administered human calcitonin at doses of 0.1-5.0 mg/kg resulted in a dose-dependent reduction in plasma calcium levels. 3. The bioavailability of intracolonically administered human calcitonin at doses of 5.0, 1.0 and 0.1 mg/kg was 0.5%, 0.9% and 0.2%, respectively. 4. Immunohistochemistry showed that human calcitonin transport across the rat colon was rapid and that a significant amount was via a transcellular pathway. 5. We conclude that human calcitonin crosses the gastrointestinal tract of rats in significant amounts and that this demonstrates the feasibility of an oral form for clinical use.


Abnormalities of ion-exchange proteins of the red cell membrane in iron deficiency anemia. The influences of iron deficiency on erythrocyte spanning membrane proteins, band 3 protein and Na(+)-K(+)-ATPase, were studied in the growing rats with iron deficient anemia. The main findings were (1) reduction of band 3 and increment of band 4.1 protein. (2) diminished rate constant of pyruvate-chloride exchange (Kp:Cl.h-1) of the erythrocytes and (3) significant decrease of Na(+)-K(+)-ATPase activity only at the early stage of iron exhaustion. In addition, there was a significant negative correlation between Kp:Cl.h-1 and Na(+)-K(+)-ATPase activity both in iron deficient rats and in the controls. It is suggested that the composition and function of the erythrocyte spanning-membrane proteins for ion exchange could be affected by iron deficiency.


[Familial adenomatous polyposis. Initial experiences with the Heidelberg polyposis register]. In order to enable consequent screening in at risk persons and life-long follow-up in patients, a computer-based polyposis registry was initiated in 1991. It now includes data of 130 FAP patients and 179 risk persons from 87 families, that were seen in our clinic since 1982. 20.5% of the "probands" (symptomatic patients) had a colorectal carcinoma at the time of diagnosis in contrast to none in the "call-up" group. The most reliable screening method as yet has proven to be the flexible sigmoidoscopy with histological verification of the adenomatous nature of the polyps. Congenital hypertrophy of the retinal pigment epithelium was only seen in 50% of our patients. The exact localisation of the FAP gene in August 1991 will soon significantly improve the prognostic value of molecular genetic screening procedures. The improved prognostic value of the method will enable early and even prenatal diagnosis. It will not any more be necessary to wait for the phenotypic manifestation (colorectal polyps) in order to be sure of the diagnosis FAP.


[Direct ileum pouch-anal anastomosis in ulcerative colitis. Technique and complications]. Fourty-nine patients (21 female, 28 male) with ulcerative colitis underwent formation of an J-ileal pouch and construction of a direct stapled pouch-anal anastomosis (IPAA) without rectal cuff. 16 patients had previously undergone surgical interventions. Overall after IPAA 7 patients (14%) experienced 11 major complications. Gastrointestinal complications included hemorrhage in 1 patient, pelvic sepsis and ileus in 3 patients, respectively. Pancreatitis and urinary infection occurred in 2 patients, sexual dysfunction in 3 patients. After closure of the ileostomy 3 patients developed late pouch-vaginal or pouch-vesical fistulas, leading to excision of the pouch. During the long-term follow-up small bowel obstruction developed in 3 patients, pouchitis in another 6 patients. After 3 months 84% of our patients were continent during daytime, 67% during nighttime. 24 months postoperatively these data concerning continence increased to 92% and 83%, respectively. We conclude that direct IPAA is a reliable procedure achieving its purpose in 96%.


Angiotensin-II acts via the type 1 receptor to inhibit 17 alpha-hydroxylase cytochrome P450 expression in ovine adrenocortical cells. In this study we have investigated the effect of angiotensin-II (A-II) on cortisol production and 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha)) expression in primary cultures of ovine adrenocortical cells and the A-II receptor subtypes that mediate these responses. While A-II alone had no stimulatory effect on cortisol secretion, it inhibited the cortisol response to ACTH (10(-8) M) in a dose-dependent manner (Ki, less than 0.1 nM; maximum inhibition, 60-80%). While prolonged treatment with ACTH (10(-8) M) increased the expression of P450(17 alpha), cotreatment with A-II (10(-8) M) also inhibited ACTH-stimulated expression, as determined by changes in mRNA, immunoreactive P450(17 alpha), and 17 alpha-hydroxylase activity. A study of the effects of the AT1 and AT2 receptor antagonists, DuP 753 and PD 123319, on binding of [125I]A-II to ovine adrenocortical cells showed that the A-II receptor population was predominantly of the AT1 subtype. The effects of A-II on inhibition of cortisol secretion in response to ACTH and the activation of phosphoinositidase-C in response to A-II alone were both fully antagonized by DuP 753, but not by PD 123319. Furthermore, the inhibitory effects of A-II on expression of P450(17 alpha), as measured at the levels of mRNA, immunoreactive protein, and enzyme activity, were reversed by DuP 753 (10(-5) M), but not PD 123319 (10(-5) M). We conclude that A-II has a potentially important role in the control of cortisol secretion and long term maintenance of P450(17 alpha) expression in the ovine adrenal cortex, and that the effects of A-II on both cortisol secretion and P450(17 alpha) expression are mediated through the AT1 receptor, which is coupled to phosphoinositidase-C.


Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle. The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in islets.


A comparison of the effects of concanavalin-A and tetradecanoylphorbol acetate on the modulation of parathyroid function by extracellular calcium and neomycin in dispersed bovine parathyroid cells. Ca2+ and other polyvalent cations as well as polycations, such as neomycin, produce similar effects on intracellular second messengers and PTH release in dispersed bovine parathyroid cells, but it is unclear whether all of these agents share the same mechanism of action. The lectin Concanavalin-A (Con-A) and the activator of protein kinase-C tetradecanoylphorbol acetate (TPA) blunt the effects of elevated extracellular calcium (Ca2+) concentrations on several aspects of parathyroid function, including PTH release, the cytosolic calcium concentration, and the accumulation of cAMP and inositol phosphates. In the present studies we used these two agents as well as pertussis toxin as probes to investigate further whether neomycin acts on parathyroid cells through the same receptor-like mechanism used by extracellular Ca2+ to regulate parathyroid function. Con-A and TPA both enhanced PTH release by about 2-fold at 0.5-1 x 10(-4) M neomycin, concentrations that inhibited PTH release to an extent (40-50%) similar to that seen with high (1.5-2 mM) Ca2+. Con-A also reduced the inhibition of agonist-stimulated cAMP accumulation by the same concentrations of neomycin. Conversely, Con-A and TPA produced 70-80% decreases in the cytosolic calcium concentration transient and the accumulation of inositol phosphates stimulated by neomycin. The effects of these two agents on neomycin-regulated parathyroid function were similar in magnitude to their actions on the modulation of these same parameters by extracellular Ca2+. Pertussis toxin, however, which we have previously shown to block the inhibitory effects of high Ca2+ and neomycin on cAMP accumulation, had no effect on the inhibition of PTH release by these two agents. These results provide further indirect evidence that polyvalent cations and polycations act on the parathyroid cell through related pathways, which probably involve cell surface moieties containing carbohydrate(s).


A 3',5'-cyclic adenosine monophosphate-dependent pathway is responsible for a rapid increase in c-fos messenger ribonucleic acid by adrenocorticotropin. ACTH rapidly and transiently increases c-fos mRNA in the rat adrenals in vivo. The present investigation was undertaken in order to determine what kind(s) of second messenger systems is involved in this increase. Rat adrenal cells were grown in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 2 days of culture, cells were treated with ACTH and various agents alone or in combination. The amount of c-fos mRNA was determined by dot blot hybridization and corticosterone levels in the media were measured by RIA. ACTH (300 pg/ml) increased c-fos mRNA transiently with a peak level after 60 min. A similar increase was observed when (Bu)2cAMP (1 mM) was substituted for ACTH. Pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase, suppressed both basal and ACTH-increased c-fos mRNA. H-89 also suppressed corticosterone production. On the other hand, neither 12-O-tetradecanoyl-phorbol-13-acetate (100 ng/ml) nor elevated potassium ion (50 mM) affected the amount of c-fos mRNA and corticosterone production. Furthermore, pretreatment with cycloheximide (5 micrograms/ml) increased both basal and ACTH-increased c-fos mRNA. These results indicate that ACTH increases c-fos mRNA by phosphorylation of preexisting trans-acting factor(s) via cAMP-dependent protein kinase in common with steroidogenesis.


Purification and characterization of guanosine 3',5'-monophosphate-inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions. We previously characterized human placental cytosolic cAMP phosphodiesterase (PDE) and found that two low K(m) cAMP PDE isoforms that were very sensitive to inhibition by cGMP and cilostamide were activated by insulin. As a first step toward understanding the mechanisms by which insulin activates this enzyme, we purified the cGMP-inhibited low K(m) cAMP PDE (cGI-PDE) from human placentas. The enzyme was purified 11,700-fold from a pool of 100,000 x g supernatant fractions of 10-15 placentas by ammonium sulfate precipitation, diethylaminoethyl-cellulose chromatography, and affinity chromatography, using an isothiocyanate derivative of cilostamide (CIT-agarose). The specific activity of the affinity-purified enzyme was 432 +/- 17 nmol/min.mg (mean +/- SD; n = 4). Gel permeation chromatography of the CIT-agarose eluates revealed one protein peak that coincided with PDE activity at an elution position of 135,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this protein peak and CIT-agarose eluates revealed the same patterns, indicating that the purified PDE preparations contained multiple proteins with apparent mol wt of 138K, 83K, 72K, 67K, 63K, and 44K. The 138K form appears to be an intact enzyme; an analogous approximately 135K form has recently been identified in rat adipocyte particulate fractions by specific immunoprecipitation or Western immunoblots. In addition, other smaller forms eluted at 135,000 daltons on gel permeation chromatography, suggesting that, although proteolyzed, they must have been associated by either noncovalent interactions or disulfide bonds. All of the protein bands observed on the sodium dodecyl sulfate-polyacrylamide electrophoresis gel reacted with rabbit antibodies raised against human platelet cGI-PDE. Ten peptides from endoproteinase Lys-C-digests of the affinity-purified placental cGI-PDE were isolated and sequenced; sequences of eight peptides were identical to the deduced amino acid sequences in the C-terminal half of a human heart cGI-PDE cDNA, while those of two peptides were not found in the heart enzyme. The sequences of the eight peptides also matched peptide sequences derived from a purified human platelet cGI-PDE. These results provide evidence that the catalytic C-terminal half domain of the placental insulin-sensitive cGI-PDE shares homology with those of human heart and platelet cGI-PDEs. K(m) and maximum velocity values for cAMP and cGMP were 0.57 microM and 862 nmol/min.mg, and 15 microM and 467 nmol/min.mg, respectively. ED50 values for cGMP, cilostamide, and Ro 20-1724 were 0.12, 0.22, and 120 microM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)


A pathway for luteinizing hormone releasing-hormone self-potentiation: cross-talk with the progesterone receptor. This study investigates the signaling pathways that lead to acute augmentation of secretagogue-induced LH secretion, the physiologically relevant manifestation of which is LHRH self-potentiation. The consequence of LHRH self-potentiation is an augmented LH secretory response to subsequent exposure to the peptide. Although the mechanism for LHRH self-potentiation remains obscure, the second messenger cAMP and the steroid hormone progesterone share common characteristics in their acute augmentation of secretagogue-induced pituitary LH secretion, suggesting that cross-talk between the peptide and steroid hormone pathways may occur. The progesterone receptor would represent a point of convergence of several effectors known to augment secretagogue-induced LH secretion. In rat anterior pituitary cells cultured in the absence of progesterone, it was found that the progesterone receptor antagonist RU486 (2 nM) inhibits LHRH self-potentiation induced by hourly pulses of 1 nM LHRH. In the absence of added progesterone, RU486 also suppresses the augmentation of LHRH-stimulated LH secretion which is a consequence of increasing [cAMP]i with either 8-bromo-cAMP (1 mM) or forskolin (1 microM) treatment. The extent of the suppression of the cAMP action in the presence of RU486 is similar to that found with the RNA synthesis inhibitor, actinomycin D. The data are consistent with the hypothesis that a LHRH-stimulated protein kinase A cascade acts, in part, through transcriptional activation of the progesterone receptor. It is concluded that the mechanism of LHRH self-potentiation requires cross-talk with the progesterone receptor.


Involvement of the Y-1 receptor subtype in the regulation of luteinizing hormone secretion by neuropeptide Y in rats. The present experiments were designed to investigate structure-function relationships, and identify the receptor subtype and postreceptor cellular mechanisms that mediate the ovarian hormone-dependent, excitatory, and inhibitory effects of neuropeptide Y (NPY) on LH release in female rats. Intracerebroventricular administration of NPY decreased plasma concentrations of LH in ovariectomized, hormonally untreated rats but stimulated LH release in ovariectomized rats pretreated with estradiol benzoate and progesterone. A similar dual response was also obtained after administration of NPY2-36. However, deletion of additional amino acids at the N terminus, as in NPY5-36, NPY11-36, NPY16-36, and NPY25-36, rendered the peptides inactive. An N-terminal fragment, NPY1-24-amide, and a discontinuous NPY analog, NPY1-4-epsilon-amino-caproic acid-25-36, similarly failed to influence LH release. The analog [Leu31,Pro34]NPY, a preferential agonist at the Y-1 NPY receptor subtype, also elicited the dual LH responses, but the preferential Y-2 receptor agonist, NPY13-36, was completely inactive. Further, only the peptides that stimulated LH release in vivo, i.e. NPY, NPY2-36, and [Leu31,Pro34]NPY, also stimulated the release of LHRH from median eminence fragments of steroid-primed rats in vitro, and the excitatory effect of [Leu31,Pro34] NPY was blocked by a noncompetitive NPY receptor antagonist, D-myo-inositol-1,2,6-trisphosphate. With the exception of NPY1-24-amide, all of the NPY fragments tested bound specifically to NPY binding sites in hypothalamic membrane preparations, but the highest binding affinities were found for the peptides that evoked biological responses in the in vivo and in vitro tests. Further analysis of the mode of action of NPY showed that the stimulation of LHRH release in vitro was unaffected by omission of Ca2+ from the incubation medium, but was prevented by two antagonists of intracellular Ca2+ mobilization, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester and ryanodine. Inhibition of prostaglandin synthesis with indomethacin blocked the stimulatory effect of the alpha 1-adrenergic agonist methoxamine on LHRH release, but not the increase produced by NPY.(ABSTRACT TRUNCATED AT 400 WORDS)


Radioimmunoassay of soluble insulin-like growth factor-II/mannose 6-phosphate receptor: developmental regulation of receptor release by rat tissues in culture. The soluble form of the insulin-like growth factor-II/mannose 6-phosphate receptor, which has been detected in serum from a variety of species, is synthesized and released by rat tissue explants in culture. Investigations into its regulation and function, however, have been hampered by inadequate means of quantification. In this paper a RIA that enables sensitive and specific quantification of soluble receptor from serum and conditioned medium is described. In order to further clarify the source of soluble receptor in serum and to determine whether release of receptor from rat tissues is under developmental regulation, receptor release from rat explants was examined and compared to serum levels of receptor. Heart, skeletal muscle, kidney, and liver explants from fetal, neonatal, weanling, and adult rats were cultured in serum-free medium, and the conditioned medium was analyzed by RIA for the presence of receptor. Soluble receptor release was highest for fetal and neonatal tissues, with weanling and adult tissues showing dramatically decreased levels. Release of soluble receptor varied between tissues, with the tissue-specific pattern altering during development, such that while heart and muscle appeared as major tissues of release in fetal animals, the liver was the tissue of highest release in adult animals. It is concluded that release of soluble receptor by rat tissues is developmentally regulated, with levels of receptor in serum reflecting the pattern of release from tissues. The RIA will prove a useful tool for further examination of the regulation and function of the soluble insulin-like growth factor-II/mannose 6-phosphate receptor.


Electrophysiological properties of a cell line of the gonadotrope lineage. The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.


Tyrosine kinase-defective insulin receptors undergo decreased endocytosis but do not affect internalization of normal endogenous insulin receptors. To characterize tyrosine kinase activity in signaling ligand/receptor internalization, metabolic labeling and surface radioligand binding were used to follow the processing of both normal and tyrosine kinase-deficient human insulin receptors. The mutant receptor (A/K1018) has an alanine substituted for lysine 1018 in the ATP-binding domain. Rat 1 fibroblasts, expressing either normal human insulin receptors (HIRc) or A/K1018 receptors, were assayed to determine the insulin receptor half-life as well as internalization and down-regulation. Our results show that insulin greatly reduces the half-life of normal insulin receptors (from 9.9 to 5.7 h). The A/K1018 receptor had a much longer half-life (24 h), which was not reduced by the presence of saturating insulin concentrations. The A/K1018 receptor does not undergo down-regulation after long term insulin exposure, while HIRc cells showed a 34% decrease in insulin receptor number. This down-regulation is accounted for by the accelerated turnover rate of normal receptors in the presence of insulin. To confirm that the kinase activity is necessary for normal endocytosis, we also show that ATP depletion in HIRc cells resulted in significant decreases in receptor internalization and that tyrosine kinase-defective receptors also fail to internalize in a different cell type (rat Fao hepatocytes). Lastly, the complement of normal rat insulin receptors in cells expressing the kinase-defective receptors endocytose normally. We conclude that the defect in endocytosis observed in kinase-defective receptors is intrinsic to this receptor and not due to a dominant inhibitory effect on cellular endocytotic machinery.


Developmental expression of rat insulin-like growth factor-II/mannose 6-phosphate receptor messenger ribonucleic acid. We have examined the developmental pattern of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor mRNA in various rat tissues from 20-day gestation fetuses and 20-day postnatal animals by Northern blotting and solution hybridization/RNase protection assays. The major mRNA species in all fetal and postnatal tissues was 9.0 kilobases. The rank order of receptor mRNA concentrations among the fetal tissues was heart greater than limb/muscle, lung, intestine, kidney, liver greater than brain, which agrees with the previously reported rank order of the tissue concentrations of receptor protein. The concentration of IGF-II/M6P receptor mRNA was significantly lower in postnatal tissues, again reflecting the relative levels of receptor protein in fetal and postnatal tissues. We measured IGF-II/M6P receptor mRNA copy number in fetal heart, the tissue with the highest concentration of receptor protein and mRNA, by including in the solution hybridization/RNase protection assay known amounts of a sense strand transcript of the receptor cDNA. This sense strand standard was quantitated by incorporating a tracer amount of [32P]UTP into the transcript and measuring the radioactivity in the product purified by gel electrophoresis. The receptor mRNA copy number in fetal heart was 74 molecules/cell. We conclude that the IGF-II/M6P receptor mRNA concentration is an important determinant of the level of receptor protein in most tissues.


Induction of prostaglandin H synthase in rat preovulatory follicles by gonadotropin-releasing hormone. Two distinct isoforms of prostaglandin (PG) endoperoxide synthase (PGS) have been identified in rat ovarian tissues: rPGSi (mol wt, 70,000-72,000) is induced by FSH and LH in preovulatory follicles, whereas the other isoform (mol wt, 69,000) is not. Induction of rPGSi is associated with LH-stimulated increases in PG biosynthesis obligatory for ovulation. Because GnRH, like LH, can also stimulate the synthesis of PGs and ovulation in the rat, this study was undertaken to determine which isoform of PGS might be induced by GnRH, in what cell type, and by what intracellular pathways. Results show that GnRH at relatively low concentrations (10(-8)-10(-7) M) induced the same isoform of PGS (rPGSi) in the same cell type (preovulatory granulosa cells) and within the same 5- to 7-h time course as did LH. Unlike LH and FSH, GnRH did not cause a major increase in cAMP, nor did GnRH induce luteinization. The effects of GnRH on rPGSi in preovulatory follicles were not mimicked by known activators of protein kinase-C (phorbol myristate acetate, bryostatin, diacyglycerol, and (+/-)ionomycin). Epidermal growth factor (but not basic fibroblast growth factor or platelet-derived growth factor), which activates a receptor-associated tyrosine kinase, caused a small increase in rPGSi. Genistein, a selective inhibitor of tyrosine kinases, blocked GnRH and LH induction of rPGSi. Taken together these results suggest that the mechanisms by which GnRH and LH selectively induce rPGSi in granulosa cells of preovulatory follicles before ovulation may converge at some step within a cellular tyrosine kinase cascade. Furthermore, the mechanisms responsible for inducing rPGSi are distinct from those required for cellular luteinization.


Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.


Evidence that folliculo-stellate cells mediate the inhibitory effect of interferon-gamma on hormone secretion in rat anterior pituitary cell cultures. Interferon-gamma (IFN-gamma) is known to inhibit the release of ACTH, PRL, and GH by rat anterior pituitary (AP) cells, stimulated by appropriate hypothalamic releasing factors. In the present study we examined the mechanisms underlying this inhibition. Dose-response studies, revealing a maximal inhibitory effect with an IFN-gamma dose as small as 10 U/ml, suggested the existence of a limiting intermediate step. In addition, in perifusion experiments with aggregates of established hormone-secreting tumor cell lines (AtT-20 and GH3), IFN-gamma had no inhibitory effect, suggesting that an accessory cell type was involved. Studies on differentially enriched cell populations of normal rat AP, obtained by velocity and buoyant density sedimentation, indicated that the inhibitory effect of IFN-gamma on stimulated ACTH and GH release was absent in those populations that contained only few folliculo-stellate (FS) cells. The presence of a minimal proportion of FS cells was found to be necessary for the inhibition to be manifest. This was seen in monolayers, but also in cultures of AP cell aggregates, which are well known to closely mimic the behavior of the AP gland in vivo. Definitive evidence for the role of FS cells was obtained by reconstitutive coculture experiments; FS cell-poor populations, which by themselves resisted the inhibitory effect of IFN-gamma, became sensitive when cocultured with an FS cell-rich population. Basal ACTH and GH release were not influenced by preincubation with IFN-gamma in either original or fractionated AP cell populations. In contrast, basal PRL release was inhibited in both systems. In cultures of AP cell populations, separated by velocity sedimentation, this inhibition showed a pattern similar to that observed for stimulated release of ACTH and GH, i.e. more inhibition in fractions with a higher proportion of FS cells. However, in FS cell-poor cultures, inhibition of basal PRL release did occur, although to a lesser degree than in FS cell-rich cultures. Our results indicate that IFN-gamma affects AP hormone secretion through the FS cell. In addition, they suggest that IFN-gamma and the FS cell constitute a system through which the pituitary gland perceives changes in the activation state of the immune system.


Characterization of oxytocin receptors in rabbit amnion involved in the production of prostaglandin E2. We characterized oxytocin (OT) receptors in purified plasma membranes from amnion, decidua, and myometrium of late pregnant rabbits using an iodinated OT antagonist (OTA). Saturation studies showed similar Kd values for specific binding sites in the 100- to 250-pM range in all three tissues. OT receptor concentrations in decidua and myometrium did not change until the day of labor (day 31), when they rose about 2.5- and 18-fold, respectively. Increases in amnion receptors were first apparent on day 28 and continued to maximal levels on day 31. There was an increase of about 230-fold from day 26 to labor, reaching 9.5 pmol/mg protein. Competition studies using analogs showed that ligand specificities of amnion and decidual membranes were indistinguishable. Those of myometrial membranes were somewhat different, possibly owing to the presence of both AVP receptors and OT receptors in the myometrium. Binding of OTA corresponded to the OT-induced release of prostaglandin E2 (PGE2) by amnion cells in culture. The effects of OT were dose dependent, agonist specific, and selectively inhibited by OTA. Amnion cells from days 22 and 28 did not respond significantly to either OT or phorbol 12-myristate 13-acetate (PMA), but cells from day 30 pregnant rabbits responded strongly to both. In contrast, calcium ionophore stimulated comparable amounts of PGE2 release from cells cultured on day 22, 28, or 30. These studies show that specific, high affinity OT receptors are associated with the release of PGE2 from rabbit amnion cells. Increases in amnion OT receptor and protein kinase-C activity precede by several days the increases in receptor concentrations in decidua and myometrium, suggesting important roles for the amnion and OT in the initiation of labor in rabbits.


Molecular size characterization of oxytocin receptors in rabbit amnion. The binding of oxytocin (OT) to receptors in rabbit amnion cells stimulates PGE2 release. We previously studied the binding characteristics, changes in receptor concentration during pregnancy, and agonist specificity of OT action on amnion cells. In this study the molecular size of OT receptors in rabbit amnion was estimated by photoaffinity labeling, radiation inactivation, and gel filtration of solubilized receptor, using an iodinated OT antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid, 2-O-methyltyrosine,4-threonine,8-ornithine,9-tyrosylamide]vasotoci n (OTA), or [3H]OT. Two electrophoretic bands, about 50 and 65 kilodaltons (kDa), were specifically covalently labeled with azidobenzoyl-[125I]OTA. Both sizes correspond to that determined by radiation inactivation, about 55 kDa, using [3H]OT binding to assess the fraction of receptor sites remaining. When we used [125I]OTA, the radiation inactivation size was about 30 kDa. These differences in radiation inactivation size suggest that the receptor binding site is comprised of more than one domain and that the binding of the antagonist involves fewer points of interaction than does OT. The molecular size of the receptor estimated from [125I]OTA binding by detergent-solubilized extracts of amnion membranes was about 350 kDa, as determined by gel filtration on columns of Sepharose 6B. Although the functional size of the receptor is about 65 kDa, it appears to be closely associated with other membrane proteins. The size estimates of amnion OT receptors agree with those in rabbit myometrium and rat mammary gland, both of which differ from amnion by contracting in response to OT. Despite different responses, OT receptors in different tissues appear to be very similar in size.


Experimental model of lead nephropathy. II. Effect of removal from lead exposure and chelation treatment with dimercaptosuccinic acid (DMSA). Male Sprague-Dawley rats were exposed to high-dose (0.5%) lead acetate for periods ranging from 1 to 9 months; then lead exposure was discontinued, and animals were sacrificed after 12 months. Controls were pair-fed. Two additional groups of low-dose (0.01%) and high-dose (0.5%) rats were exposed to lead for 6 months, then lead was discontinued and the rats were treated with three 5-day courses of 0.5% DMSA (dimercaptosuccinic acid) over the next 6 months. Controls were rats exposed to lead for 6 months, then removed from exposure for 6 months without receiving DMSA. Low-dose lead-treated rats showed no significant pathological changes with or without DMSA treatment, but exhibited a significant increase in GFR after DMSA. High-dose lead-treated animals showed no functional or pathological changes when lead exposure was discontinued after 1 month. However, when duration of exposure was 6 or 9 months, GFR was decreased and serum creatinine and urea nitrogen were increased as compared to controls. Tubulointerstitial disease was severe. Administration of DMSA resulted in an improvement in GFR and a decrease in albuminuria, together with a reduction in size and number of nuclear inclusion bodies in proximal tubules. However, tubulointerstitial scarring was only minimally reduced. It may be concluded that, except for brief initial exposure, discontinuation of high-dose lead exposure fails to reverse lead-induced renal damage. Treatment with the chelator, DMSA, improves renal function but has less effect on pathological alterations. As GFR improved after DMSA treatment in both low-dose and high-dose lead-treated rats, irrespective of the degree of pathological alterations, it may be concluded that the DMSA effect is most likely mediated by hemodynamic changes.


Tumor incidence was not related to the thickness of visceral pleural in female Syrian hamsters intratracheally administered amphibole asbestos or manmade fibers. Histological observations were performed on female Syrian hamsters 2 years after the intratracheal administration of amphibole asbestos, amosite, and crocidolite to evaluate the tumorigenicity of six types of fine manmade fibers (reported previously). A mesothelioma and a lung tumor were induced in 20 animals administered amosite, but no tumors were found in the crocidolite group. Because this incidence is not higher than that of manmade fibers, such as basic magnesium sulfate fiber [9 tumor-bearing hamsters in 20 hamsters (9/20)], metaphosphate fiber (5/20), calcium sulfate fiber (3/20), and fiberglass (2/20), it is suggested that some types of manmade fibers have a greater ability than asbestos to induce tumors. Moreover, as a specific observation in manmade fiber groups, tumors were induced at intracelial organs rather than at the pleural cavity. On the other hand, the average thickness of visceral pleura was higher in all asbestos and manmade fiber groups than in the control (2.9 microns), for instance, 36.95 microns in potassium titanate fiber group, 15.90 microns crocidolite group, 13.00 microns basic magnesium sulfate fiber group, and 10.45 microns in the rockwool group. Although both pleural thickening and mesothelioma are known as peculiar lesions in asbestos-exposed people, it might also be suggested that these lesions could be induced by different mechanisms from the result that there was no relation between the pleural thickening and mesothelioma incidence in hamsters.


Deposition, clearance, and translocation of chrysotile asbestos from peripheral and central regions of the rat lung. We investigated the pulmonary deposition, clearance, and translocation of chrysotile asbestos in the context of our previously developed model of asbestosis in the rat. Adult male rats were exposed for 3 hr to an aerosol of chrysotile asbestos. Subgroups were sacrificed up to 29 days postexposure and the lungs of the animals fixed. Peripheral and central regions of the left lung were resected, digested, and analyzed for fiber content by scanning electron microscopy. Pulmonary deposition did not differ between peripheral and central regions. There was no evidence of translocation of fibers from central to peripheral regions. The average diameter of retained fibers decreased over time, consistent with longitudinal splitting. The average length of retained fibers increased over time, consistent with slower clearance of longer fibers. We employed a novel counting scheme to ensure accurate fiber number measurements, allowing the calculation of clearance rates for fibers 0.5 to greater than or equal to 16 microns in length. Fibers of length greater than or equal to 16 microns were cleared slowly, if at all. These findings could have important implications for the pathogenesis of asbestos-related pleural disease. Many fibers are deposited in the peripheral region, and the longest (greater than or equal to 16 microns) will persist there for extended periods.


Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen-binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein residing in the endoplasmic reticulum, and is assumed to be a molecular chaperone specific to collagen. Two-dimensional gel electrophoresis and immunoprecipitation studies showed that the expression of HSP47 was significantly induced during the differentiation of mouse teratocarcinoma F9 cells by treatment with retinoic acid alone or with retinoic acid and dibutyryladenosine 3',5'-phosphate. The induction of type-IV collagen was also observed during F9-cell differentiation. For further analysis, we cloned cDNA encoding mouse HSP47 from a cDNA library of BALB/c 3T3 cells and performed Northern-blot analysis. The cDNA contained a signal sequence at the N-terminus and an endoplasmic-reticulum-retention signal, RDEL, at the C-terminus. An homology search revealed that mouse HSP47, as well as chick HSP47, belonged to the serine protease inhibitor superfamily. While chick HSP47 mRNA was 4.5 kb with a long (2-kb) 3' untranslated region, mouse and human HSP47 mRNA were 2.5 kb, with a 0.8-kb 3' untranslated region. Northern-blot analysis revealed that the concurrent induction of HSP47 and type-IV collagen during F9-cell differentiation, and the transient induction of HSP47 after heat shock was regulated at the level of mRNA accumulation. These results suggested that HSP47 was closely related to collagens in terms of its expression as well as in its functional relevance.


Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI. The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.


Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms. A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.


Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase. Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475-480], yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second Pi is faster for the three-metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase-activity regulation.


Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms. It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.


Spontaneously occurring staphylococcal mutants resistant to clinically achievable concentrations of ciprofloxacin and temafloxacin. The frequency of spontaneously occurring mutants resistant to 0.5, 1, 2, 4 and 8 micrograms of temafloxacin or ciprofloxacin per milliliter was documented with four Staphylococcus aureus and four Staphylococcus epidermidis strains. Resistant mutants were recovered at two- to four-fold the MIC of either drug, and they displayed cross-resistance to other fluoroquinolones. Resistance to temafloxacin occurred at frequencies lower than those observed with equal concentrations of ciprofloxacin. Resistance to greater than four-fold the MIC of either drug was not detected (frequencies less than 10(-10).


Detection of JC virus by polymerase chain reaction in cerebrospinal fluid from two patients with progressive multifocal leukoencephalopathy. The polymerase chain reaction (PCR) was used to identify JC virus (JCV) in the cerebrospinal fluid of two patients with progressive multifocal leukoencephalopathy confirmed by brain biopsy. In addition, JCV viremia was demonstrated by PCR in one case. JCV detection in spinal fluid by PCR may be the first non-invasive technique available for the diagnostic confirmation of progressive multifocal leukoencephalopathy.


In vitro activity of azithromycin and tetracycline against 358 clinical isolates of Brucella melitensis. The in vitro activity of azithromycin against human pathogenic strains of Brucella melitensis was tested at three centres and compared to that of tetracycline, the standard antibiotic currently used for the treatment of human brucellosis. MIC determination was carried out. Tested concentration ranges for both azithromycin and tetracycline were between 0.03 and 16 micrograms/ml. Brucella melitensis biotype 1, strain M16 was employed as a control microorganism. A total of 358 Brucella melitensis strains from human blood cultures were tested. MIC90 (micrograms/ml) values ranged from 0.5 to 1.00 for azithromycin and were 0.25 for tetracycline. It was concluded that there was little difference in the sensitivity of pathogenic Brucella melitensis to azithromycin and tetracycline isolated from three different regions in Spain. These results encourage further investigations on the possible therapeutic role of azithromycin in brucellosis.


Increased ability of murine bone marrow stromal cells immortalized with simian virus 40 (SV40) to retain haemopoietic cells and support long-term haemopoiesis in vitro. A simple method for short-term recharge of SV40-immortalized marrow stromal cell (MSC) lines based on their specific interaction with the appropriate haemopoietic cells is described. During the first week after the recharge the production of polymorphonuclear and mononuclear blood cells is an order of magnitude higher than in the recharged primary MSC cultures. Progenitor-derived granulopoiesis is predominant. The long-term repopulating potential of the haemopoietic stem cells (HSC) in the recharged cultures is demonstrated when they are cultured in tissue culture wells (Sutherland et al., 1990). The blood cells produced in the later passages show signs of malignancy.


Biology of Rous sarcoma virus adapted for replication and transformation of duck cells. A duck-adapted variant of the Prague strain of Rous sarcoma virus and its molecularly cloned derivatives were characterized biologically. The virus replicated and transformed with the same efficiency both duck and chicken embryo fibroblasts. The daPR-RSV-C was also used for induction of tumours in newborn hamsters. The virus from virogenic hamster tumours could be rescued using the duck cells as indicator cells. The virus rescue was influenced by restricted replication of the virus in the indicator cells.


Anticipation and early detection can reduce bowel elimination complications. Adequate bowel elimination is essential for physiologic functioning and daily comfort of older patients. A careful assessment of normal bowel elimination patterns will help to prevent unnecessary bowel problems when the older patient is hospitalized or admitted to a long-term care facility. Constipation and incontinence are the two most common bowel elimination problems affecting older adults. Many simple nursing interventions exist that will help to prevent major complications that can occur when constipation or incontinence is present.


[Effects of lithium and antidepressants on monoaminergic receptors and receptor-coupled adenylate cyclase system in rat brain]. The mechanisms of action of lithium and antidepressants were investigated with reference to effects of these drugs on monoaminergic receptors and receptor-coupled adenylate cyclase systems in rat brain. Oral administration of lithium carbonate for 21 days decreased significantly the density of beta-adrenergic receptors in rat cerebral cortex, which is the same change as reported as the result of long-term treatment with many antidepressants. With regard to 5-hydroxytryptamine (5-HT) receptor subtypes, lithium treatment reduced the maximum number of 5-HT1A receptors in rat hippocampus but not in cerebral cortex, whereas repetitive injections with imipramine or desipramine did not. beta-Adrenoceptor-coupled adenylate cyclase activity was subsensitized by long-term lithium treatment in consistency with above-mentioned down-regulation of beta-adrenergic receptors. Stimulation of adenylate cyclase activity by non-hydrolyzable GTP analogue, guanyl-5'-ylimidodiphosphate (Gpp(NH)p), was, however, unaltered in lithium-treated rats as compared with controls. On the other hand, 5-HT1A-mediated inhibition of forskolin-stimulated adenylate cyclase in rat hippocampal membranes was not altered by chronic treatment with lithium or antidepressants. Gpp(NH)p-induced inhibition of forskolin-stimulated adenylate cyclase activity was not influenced by lithium treatment, either. [3H]Forskolin binding to rat cerebral cortex, which is assumed to be associated with the activated complex of catalytic subunit of adenylate cyclase and stimulatory guanine nucleotide-binding regulatory proteins (Gs), was not changed by administration of lithium or antidepressants under any condition studied. Pertussis toxin (islet-activating protein, IAP) sensitive G proteins (Gi/Go) as determined by using IAP-catalyzed [32P]ADP-ribosylation was not altered by lithium- or antidepressant-treatment, either. The implication of these results is discussed with a view of clarifying the mechanisms of action of these thymoleptic drugs.


Nutrient interaction in relation to glycaemic and insulinaemic response. Although it is known that protein, fat and fibre reduce the postprandial glycaemia following an oral carbohydrate load, the nature and extent of interaction of different nutrients with one another in this respect is not well understood. The present study was designed to explore systematically the glycaemic and insulinaemic response to glucose (G) alone, or in combination with one or more of the following: casein (CS), maize oil (MO), cellulose (CL) and pectin (P). Besides 100 g G, eleven isoenergetic and six isocarbohydrate meals were studied on healthy adult males using an incomplete block design. Addition of other nutrients to G led to a lowering of the glycaemic response. The lowest glycaemic responses were seen in case of meals containing the largest number of nutrients. P was more effective in reducing postprandial glycaemia than CL. As in case of glycaemic response, low insulinaemic responses were also associated with P-containing meals, and meals containing the largest number of nutrients. But unlike in case of glycaemic response, there was a tendency for elevation of the insulinaemic response in case of CL-containing meals. The degree of attenuation of glycaemic response observed with meals containing several nutrients was roughly predictable on the basis of the attenuation observed with meals in which only one nutrient had been added at a time to G. But the glycaemic response of natural foods is unlikely to be predictable on the basis of their nutrient composition because of the overriding influence of several other factors such as physical form, cooking, processing, storage and antinutrient content of the food.


Pulmonary function studies in 15 to 18 years age workers exposed to dust in industry. Pulmonary function tests (VC, FEV1% and FEF25-75%) were evaluated in 15 to 18 years age workers employed in slate pencil industry exposed to silica dust, in wool carpet industry exposed to wool dust and in diamond cutting and polishing exposed to carbon dust. These values were compared with the values obtained in clinically healthy non-smokers of the same age group. The results revealed significant impairment of VC in diamond workers, and FEF25-75% in slate pencil workers. When the values were observed according to smoking habits in diamond workers, VC, FEV1 and FEF25-75% were all significantly reduced in smokers whereas in non-smokers only VC was lowered significantly. Among slate pencil workers FEF25-75% was significantly reduced in both smokers and non-smokers. Wool dust exposed workers showed reduced values than normal subjects. The detailed results including the prevalence of various pulmonary impairments were presented.


Increased food intake following carotid and systemic insulin infusions. In order to determine whether insulin receptors in the brain or in the periphery are more important for the control of food intake, four doses of insulin (0.1, 0.5, 1.0 and 2.0 U/day) were each infused continuously over 24 h into rats with chronic catheters in the common carotid artery (C) or the superior vena cava (VC). Daily food intake was unchanged from baseline levels by insulin doses of 0.1 to 1.0U. However, intake increased by 8.1 +/- 1.3 kcal for the C rats and by 10.2 +/- 1.8 kcal for the VC rats during the 2.0U infusion and was also increased on the subsequent day, by 10.9 +/- 1.9 kcal and 15.4 +/- 3.0 kcal for C and VC groups respectively. Rats were fed half an hour after insulin infusions began and measures of short-term intake made over the next 30 min were unaffected by any of the insulin doses. When injected into C catheters prior to sacrifice greater than 90% of 57Co labelled microspheres were recovered in arterioles of the brain, skull and facial regions. As brain blood flow is 1.8% of cardiac output, insulin levels in the brain blood supply would have been substantially greater for C rats. The similar increases in food intake for C and VC rats clearly show that insulin fails to produce greater effects when infused directly into the brain. Instead, these findings suggest that the effect of insulin on daily food intake is predominantly a function of insulin receptor activation in the periphery.


Familial resemblance of body fat distribution: the Minneapolis Children's Blood Pressure Study. Although many reports have shown that obesity (as defined by weight for height indices) tends to run in families, considerably fewer data are available concerning the familial resemblance of body fat distribution as defined by the waist to hip ratio (WHR), which is a risk factor for coronary heart disease. This question was examined among 712 participants of the Minneapolis Children's Blood Pressure Study. For each family member separately (son, daughter, father and mother) the distribution of the WHR was divided into quintiles. Quintiles of WHR were cross-classified between child and parent to examine the proportion of subjects clustering in each quintile. A significantly higher than expected proportion of parent-child WHRs clustered in the top quintile. For example, 42.5% of sones were placed in the top quintile of their father's WHR (P less than 0.001) and 36.7% of daughters were placed in the top quintile of their mother's WHR (P less than 0.001). Multiple linear regression analyses revealed that these findings were independent of parent and child body mass indices and other covariates. These results indicate significant familial aggregation of body fat distribution and may aid in suggesting specific primary preventive strategies targetted at appropriate families to reduce the risk of cardiovascular disease.


Prospective study of the influence of social factors in childhood on risk of overweight in young adulthood. The purpose of the study was to assess prospectively the influence of social factors in childhood on risk of overweight in young adulthood, while taking into account degree of fatness in childhood. A random selection of children 9-10 years of age attending the third school grade in Copenhagen schools in 1974 was performed. Parental school education, householder's occupational status, and quality of dwellings in the rearing area were recorded. Ten years later a follow-up was carried out, and 756 (86%) of the 881 eligible individuals participated. The effect of the social factors on the risk of overweight in adulthood was analysed by logistic regression analysis, including gender and degree of fatness in childhood as covariates. The relationship between the traditional socio-demographic factors (parental education and occupational) and risk of overweight in young adulthood was insignificant, when controlling for degree of fatness in childhood and gender. However, the risk of overweight was highly increased for individuals reared in an area with poor quality of dwellings compared to those from a good area (P less than 0.001), even when controlling for the effect of parental education and occupation (P less than 0.001). In conclusion, rearing area had a much stronger influence on risk of overweight in young adulthood than parental education and occupation.