Enhancement of cAMP degradation by increased cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) activity is thought to be an important component of the mechanism whereby insulin counteracts catecholamine-induced lipolysis in adipocytes. In this study the selective cGI-PDE inhibitor OPC3911 was used to evaluate this role of cGI-PDE activation in intact rat adipocytes with special reference to changes in cAMP levels measured as cAMP-dependent protein kinase (cAMP-PK) activity ratios. OPC3911 completely blocked (IC50 = 0.3 microM) the maximal inhibitory effect of insulin on noradrenaline-induced lipolysis and the net dephosphorylation of hormone-sensitive lipase and other intracellular target proteins for insulin action, whereas insulin-induced lipogenesis was not changed. The effect of OPC3911 on cAMP-PK activity ratios at different levels of lipolysis achieved by noradrenaline stimulation revealed that the reduction of cAMP-PK caused by 1 nM insulin was completely blocked by 3 microM OPC3911. The effect of OPC3911 was not due to an excessive increase in cellular cAMP resulting in 'supramaximal' lipolysis unresponsive to insulin. These data demonstrate that reduction in cAMP levels by the activation of cGI-PDE may be sufficient to account for the antilipolytic action of insulin.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Ontology-based configuration of problem-solving methods and generation of knowledge-acquisition tools: application of PROTEGE-II to protocol-based decision support.

PROTEGE-II is a suite of tools and a methodology for building knowledge-based systems and domain-specific knowledge-acquisition tools. In this paper, we show how PROTEGE-II can be applied to the task of providing protocol-based decision support in the domain of treating HIV-infected patients. To apply PROTEGE-II, (1) we construct a decomposable problem-solving method called episodic skeletal-plan refinement, (2) we build an application ontology that consists of the terms and relations in the domain, and of method-specific distinctions not already captured in the domain terms, and (3) we specify mapping relations that link terms from the application ontology to the domain-independent terms used in the problem-solving method. From the application ontology, we automatically generate a domain-specific knowledge-acquisition tool that is custom-tailored for the application. The knowledge-acquisition tool is used for the creation and maintenance of domain knowledge used by the problem-solving method. The general goal of the PROTEGE-II approach is to produce systems and components that are reusable and easily maintained. This is the rationale for constructing ontologies and problem-solving methods that can be composed from a set of smaller-grained methods and mechanisms. This is also why we tightly couple the knowledge-acquisition tools to the application ontology that specifies the domain terms used in the problem-solving systems. Although our evaluation is still preliminary, for the application task of providing protocol-based decision support, we show that these goals of reusability and easy maintenance can be achieved. We discuss design decisions and the tradeoffs that have to be made in the development of the system.

Artificial Intelligence↗

PubMed1995

A novel co-stimulatory T cell antigen co-expressed on renal cell carcinoma.

The A6H mAb raised primarily against human renal cell carcinoma (RCC) has previously been shown to bind strongly to RCC, to some degree to colon carcinoma but only marginally to a variety of normal tissues. Immunohistochemical analysis or RCC tissues containing tumor-infiltrating lymphocytes revealed that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H mAb stained 85-90% of both CD4+ and CD8+ T cells, but not granulocytes, monocytes, NK cells or B cells. Furthermore, 85-90% of naive and memory T helper cells were stained with A6H suggesting that the A6H mAb defines unique subsets within these T cell populations. Dual staining showed that A6H mAb bind to an antigen that is clearly distinct from other cell surface molecules on T cells, including CD28, CD29, CD26, CD44 and ICAM-2. A6H mAb binding induced a second signal in anti-CD3 mAb activated T cells, resulting in cell proliferation, IL-2 receptor expression and vigorous production of IFN-gamma and TNF, and production of minor amounts of IL-2. Immunoprecipitation with A6H mAb indicated a molecular weight of 120-140 kDa on both T cells and RCC. We suggest that the A6H mAb defines a unique T cell surface antigen which is involved in signal transduction and is expressed on subsets of human T cells. The co-expression of A6H on T cells and tumor cells suggests a possible function related to common properties of these cells.

Antibodies, Monoclonal↗

PubMed1995

Heart failure: a growing public health problem.

At a time when deaths from coronary heart disease and stroke are markedly declining, mortality from heart failure is increasing. Heart failure is a costly and devastating disease, and throughout much of the industrialized world, escalating health-care costs constitute a serious burden on both public and private systems of financing health care, and about one-third of all heart failure patients are admitted to hospital each year. Both prevalance and incidence of heart failure increase steeply with increasing age. The prevalence rate is about 1% at the age of 50, whilst at the age of 80 and above, almost one out of 10 persons will suffer from heart failure. Until recently, the goals for heart failure treatment were to relieve symptoms and enhance functional capacity. Recently, some large scale studies have shown that ACE inhibitors can reduce mortality, prevent development of heart failure, avoid the need for hospitalization and improve prognosis. ACE inhibitors may therefore have promising effects both on patients and on society. A challenge for the future must be early recognition and timely and adequate treatment of heart failure. Such a strategy might have great economic benefits as far as public health is concerned. However, the most rewarding efforts for the population will be to prevent the underlying causes of coronary heart failure as well as risk factors for heart failure. This review will study the magnitude of heart failure as a growing public health problem, the underlying causes, risk factors and treatment.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1995

Recovery from sevoflurane and isoflurane anaesthesia after outpatient gynaecological laparoscopy.

As the low blood solubility (blood gas partition coefficient 0.69) of sevoflurane suggests a rapid emergence from anaesthesia, recovery from sevoflurane anaesthesia was compared to isoflurane in outpatient gynaecological laparoscopy. Fifty ASA I or II, consenting women participated in a randomised, controlled and single blind study. The patients received, after induction of anaesthesia with propofol, either sevoflurane or isoflurane, both with 67% nitrous oxide in oxygen, for maintenance of anaesthesia. The study drug was administered at 1 MAC (end tidal concentration 0.6% for sevoflurane and 0.5% for isoflurane) but adjusted in 0.5 MAC steps, if clinically indicated. Before the end of surgery the end tidal concentration of the study drug was reduced to 0.5 MAC. Recovery assessments were made from the time anaesthetic gases were discontinued. The subjects were able to open eyes in 2.3 (0.8-7.0) min and 4.1 (2.0-6.8) min, orientate in 2.8 (1.0-6.8) min and 4.7 (2.2-8.3) min and follow orders in 2.6 (0.7-6.8) min and 4.3 (1.2-7.3) min, in the sevoflurane and isoflurane groups, respectively (P < 0.05) [median (range)]. Walking was achieved in 72 (24-464) min and 66 (35-134) min, tolerance of oral fluids in 37 (15-88) min and 35 (45-161) min and voiding in 262 (96-459) min and 217 (52-591) min in the sevoflurane and isoflurane groups, respectively (NS). Overall home readiness was achieved in 281 (96-708) min after sevoflurane group and 242 (96-591) min after isoflurane (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1995

Comparison of eltanolone and thiopental in anaesthesia for termination of pregnancy.

Eltanolone, a new steroid hypnotic, was compared to thiopental in short anaesthesia. Sixty unpremedicated, Asa 1-2 women were randomized to receive eltanolone 0.6 (group E1) or 0.8 mg.kg-1 (group E2) or thiopental 4 mg.kg-1 (group T) for induction of anaesthesia. One minute before induction glycopyrrolate 0.2 mg and alfentanil 15 micrograms.kg-1 i.v. were administered. If induction failed, additional boluses of the test drug were given. Anaesthesia was maintained with 67% nitrous oxide in oxygen, and additional bolus doses of the test drug were given in a standardized fashion, if needed. Recovery was assessed by a research nurse blinded to the agent used. Mean +/- s.d. induction doss were 0.7 +/- 0.1 mg.kg-1 (group E1), 0.8 +/- 0.1 mg.kg-1 (group E2) and 4.0 +/- 0.0 mg.kg-1 (group T). Induction time was prolonged in the group E1 compared to the other two groups (P < 0.05). Side effects were few in all groups, but involuntary muscle movements or hypertonus occurred in 23% of the patients in the eltanolone and in 3% in the thiopental groups (n.s). Early recovery (eye opening, orientation and sitting) was slower in both eltanolone groups compared to the thiopental group (P < 0.01). No differences between the groups were found in toleration of oral fluids, walking, voiding, postoperative analgesic requirements or postoperative nausea and vomiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Induced↗

PubMed1995

Tyrosine phosphorylation of the growth hormone (GH) receptor and Janus tyrosine kinase-2 is involved in the insulin-like actions of GH in primary rat adipocytes.

The nature of tyrosine phosphorylations induced by GH in relation to the insulin-like metabolic effects in primary rat adipocytes was investigated. Unlike other cells, e.g. 3T3-F442A fibroblast, in which GH is believed to initiate cell differentiation through activation of the Janus tyrosine kinase-2 (JAK2), the adipocytes are metabolically active and fully differentiated cells that do not proliferate. Thus, it cannot be assumed that the same molecular mechanisms relay the acute insulin-like effects of GH. In adipocytes responsive to these effects, we found that GH induced tyrosine phosphorylation of a 114-kilodalton membrane protein, identified as the GH receptor, and a 130-kilodalton cytosolic protein, identified as JAK2. In contrast, these phosphorylations were not seen in adipocytes refractory to these effects of GH. The GH concentration dependency (ED50,1-2 nM) of these phosphorylations coincided with the increase in lipogenesis and the decrease in noradrenaline-induced lipolysis caused by the hormone. In analogy with the effects of insulin, the onset of phosphorylation was rapid (t1/2, < 1 min) and preceded the metabolic responses. The observations that a small fraction of the receptor pool became tyrosine phosphorylated and that the level of phosphorylation induced by GH decreased at higher GH concentrations agree with the concept that GH-induced dimerization of the receptor is necessary for signal transduction. We conclude that tyrosine phosphorylation of JAK2 and the GH receptor seems to be involved in the signal transduction mechanism leading to the insulin-like effects of GH in adipocytes. Importantly, the signal pathways for GH and insulin clearly differ at the receptor level, but seem to converge at or before the level of insulin receptor substrate 1 or 2 phosphorylation that has been shown to occur in response to both of these hormones.

Adipocytes↗

PubMed1995

Inhibition of activation of human T lymphocytes by the complement C1 esterase inhibitor.

The major histocompatibility complex (MHC) class I molecules have been shown to be substrates to both of the complement C1 esterases. The effect of a C1 esterase-mediated cleavage of the MHC class I molecules on the activation process of lymphocytes was investigated by including the complement C1 esterase inhibitor (C1-Inh) in the medium during activation of human peripheral lymphocytes by staphylococcal enterotoxin A (SEA). The C1-Inh was shown to inhibit the activation of both CD4+ and CD8+ cells. No effect on activation of B lymphocytes was recorded, although the complement C1 complex was shown to bind to the B lymphocytes. Furthermore, the C1 complex bound to mononuclear cells was shown to be cleaved into molecular weights corresponding to the activated forms of the C1 esterases. The effect of the C1-Inh was much more pronounced at low cell density and the inhibition was not affected by the addition of interleukin-2 (IL-2). However, the inhibition was reduced when the cells were disturbed by addition of new portions of C1-Inh, 24 and 48 hr after the initiation of the activation. This indicates that the C1-Inh interference with the activation of T lymphocytes is mediated through a mechanism that requires some form of cell contact.

Complement C1↗

PubMed1994

Expression of a neutral horseradish peroxidase in Escherichia coli.

A cDNA sequence encoding a neutral horseradish peroxidase, HRP-n, was found to be growth inhibiting and under certain circumstances also toxic to Escherichia coli upon expression. The growth inhibiting and toxic activity was identified to a sequence of 192 nucleotides which encode the first deduced 64 N-terminal amino acids of the total 299 amino acids in the mature, neutral horseradish peroxidase. The sequence makes part of the active site of the enzyme and is very conserved among peroxidases. The cDNA sequence was cloned in the heat inducible expression vector pJLA603. The toxic effect was mediated by the produced polypeptide since no growth inhibiting or toxic activity was observed when the cDNA sequence was induced after ligation in a wrong reading frame. Generation of oxygen radicals was not the mechanism behind the toxicity, since the effect was observed even after induction of the complete HRP-n sequence or the identified toxic sequence under anaerobic conditions.

Amino Acid Sequence↗

PubMed1994

Purification of proteins by the use of hydrophobic zeolite Y.

Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH4)2SO4 or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be absorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH4)2SO4 fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5.

Adsorption↗

PubMed1994

Estrogen regulation of the estrogen receptor and insulinlike growth factor-I in the rat uterus: a potential coupling between effects of estrogen and IGF-I.

The interrelationship between estrogen and insulin-like growth factor-I (IGF-I) in the regulation of uterine growth was studied in the rat. The levels of the estrogen receptor (ER), ER mRNA, and IGF-I mRNA in rat uterus and liver were monitored. Uterine ER in normal cycling rats was highest in proestrus and diestrus, as was IGF-I mRNA. ER mRNA and plasma estradiol peaked in proestrus. Hepatic ER mRNA and IGF-I mRNA were highest in diestrus, whereas ER was not significantly changed during the estrous cycle. The temporal effects of multiple injections or continuous infusion of 17 beta-estradiol in ovariectomized rats were examined. In the uterus of animals subjected to multiple injections, a 10-fold increase in IGF-I mRNA was seen 24 h after the start of the treatment, whereas rats given continuous infusion of estradiol showed a more than 16-fold increase. In both groups, the increase of IGF-I mRNA was transient although estrogen treatment was continued. To study local hormonal effects, ovariectomized rats were given estradiol in vaginal implants. The uterine IGF-I mRNA level increased two-fold in 3 days. The ER mRNA level increased 1.5-fold and the uterine weights were doubled. The plasma estradiol concentration did not change during the treatment. A separate experiment was carried out to establish whether IGF-I itself exercises estrogen-like effects. Ovariectomized rats were given hrIGF-I in osmotic minipumps for 3 days. The uteri of the treated animals weighted significantly more than did the controls. Quantitation of the level of uterine estrogen receptors revealed a significant decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Androgen regulation of the insulin-like growth factor-I and the estrogen receptor in rat uterus and liver.

For the first time testosterone is shown to be an important regulator of the insulin-like growth factor-I (IGF-I) in the rat uterus under in vivo conditions. In this study the regulation of IGF-I and the estrogen receptor (ER) by gonadal steroids in the uterus and liver of female rats was monitored. The ER level was assayed by hormone binding after treatment with testosterone, 5 alpha-dihydrotestosterone or estradiol and specific mRNA species were analyzed by a solution hybridization/RNase protection assay using 35S-labeled RNA probes. Ovariectomized rats restored uterine weight after treatment with testosterone. Uterine IGF-I mRNA was more than 20-fold higher in testosterone treated rats compared to untreated ovariectomized controls after 48 h treatment. The effects of testosterone on ovariectomized animals was followed in a timecourse study. Testosterone administration increased uterine IGF-I mRNA expression during the first 48 h and the maximally induced level was maintained throughout the duration of the experiment (168 h). Since induction of IGF-I mRNA by estrogen is transient, these data indicate that androgen and estrogen increase IGF-I mRNA by different mechanisms. Regulation of IGF-I mRNA by gonadal steroids was also studied in hypophysectomized animals. The rats were given either testosterone, 5 alpha-dihydrotestosterone or estradiol, and uterine IGF-I mRNA was measured after 1 week of treatment. At this timepoint estrogen treated rats showed levels of IGF-I mRNA not significantly different from those of hypophysectomized controls. In contrast testosterone and 5 alpha-dihydrotestosterone increased the IGF-I mRNA level 30 and 40 times, respectively, relative to hypophysectomized control animals. Since 5 alpha-dihydrotestosterone is not convertable to estrogen, the induction by testosterone was considered to be a true androgenic phenomenon.

Androgens↗

PubMed1994

Can laboratory testing improve screening strategies for deep vein thrombosis at an emergency unit?

OBJECTIVES: To study various markers of blood coagulation and fibrinolysis in relation to the extension of deep vein thrombosis (DVT), and to compare the diagnostic usefulness of these markers as screening tests for excluding DVT. DESIGN: A clinical study of patients admitted to an emergency unit. SETTING: Ostra Hospital, Göteborg, Sweden. SUBJECTS: One hundred and five patients with a clinical suspicion of DVT. MAIN OUTCOME MEASURES: Phlebography was used as the reference method for a diagnosis of DVT. Small distal thromboses as well as large proximal thromboses were included. Plasma D-dimer as well as other markers of coagulation and fibrinolysis were analysed. RESULTS: Twenty-eight proximal and 20 distal DVTs were found. Plasma D-dimers (one ELISA and two latex assays), fibrin monomer, prothrombin fragment 1 + 2 (F1+2), thrombin-antithrombin III complex (TAT) and the t-PA-PAI-1 complex were all significantly correlated to the extension of DVT, whilst fibronectin, tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scru-PA) and plasminogen activator inhibitor 1 (PAI-1) were not. The sensitivity was 94% for the D-dimer ELISA and one of the latex methods (latex-B), at a specificity of 60% and 68%, respectively. The negative predictive value was 92% for ELISA and 93% for latex-B, and both assays showed a negative predictive value of 100% for proximal DVTs. Fibrin monomer, F1+2, TAT, D-dimer (latex-S) and the t-PA-PAI-1 complex all showed lower negative predictive values (88, 84, 79, 78 and 65% respectively). CONCLUSIONS: Sensitivity and negative predictive values for a latex assay (D-dimer latex-B) was similar to that of a D-dimer ELISA: With a sensitivity of 94% (100% for proximal DVTs) such a latex assay may be included in a screening strategy for DVT at an emergency unit. However, the safety of such an approach has to be tested in other prospective studies.

Adult↗

PubMed1994

The hormonal regulation of the oestrogen receptor in rat liver: an interplay involving growth hormone, thyroid hormones and glucocorticoids.

The regulation of the formation of the hepatic oestrogen receptor (ER) in adult female rats was studied by assaying steady state levels of ER and ER messenger RNA under different endocrine conditions. Hypophysectomy (HX) drastically reduced ER levels from 67.5 +/- 7.9 to 8.4 +/- 0.5 (means +/- S.E.M.) fmol/mg cytosolic protein. Continuous infusion of growth hormone (GH) to HX animals tripled ER and doubled ER mRNA levels. Treatment with triiodothyronine (T3) in a high dose (10 micrograms/day) doubled ER mRNA levels. The effects of T3 were dose-dependent, since a lower dose (1 microgram/day) increased neither ER nor ER mRNA levels. ER mRNA concentrations were increased by GH to 481 +/- 44% and by T3 to 372 +/- 35% of HX control levels 4 h after single injections of the hormones in HX animals. The glucocorticoid dexamethasone (DEX) alone increased neither ER nor ER mRNA levels in HX animals. DEX and GH in combination increased ER 5-fold and ER mRNA 2-fold compared with control levels in HX animals, whereas DEX and T3 in combination increased neither ER nor ER mRNA levels. Treatment with prolactin affected neither ER nor ER mRNA levels in HX rats. Insulin-like growth factor I (IGF-I) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured. GAPDH mRNA levels were increased 2.5-fold in HX rats by DEX and T3 in combination and almost 2-fold by DEX and GH in combination. IGF-I mRNA levels in HX rats were increased 4.5-fold by continuous infusion of GH alone, 6-fold by GH and T3 in combination, and 2.5-fold by GH and DEX in combination. These data indicate that both GH and T3 act directly on the liver to increase ER mRNA levels. GH, the most important of these hormones, also acts at the translational and/or post-translational level to increase ER protein levels. DEX treatment suppresses the stimulatory effects of T3, but not of GH.

Animals↗