Short sleep duration as a possible cause of obesity: critical analysis of the epidemiological evidence.

Systematic literature search for epidemiological evidence for an association of short sleep with weight gain and eventual development of obesity provided 71 original studies and seven reviews of various subsets of these studies. We have summarized the evidence for such an association with particular emphasis on prospective studies. The studies showed that short sleep duration is consistently associated with development of obesity in children and young adults, but not consistently so in older adults. We have identified critical aspects of the evidence, and assessed the possibility for interpretation of the evidence in terms of causality. We have discussed the requirement of temporal sequence between putative exposure and outcome and the implications of the time lag between them, the problems in adequate measurements of exposure and effects, the possible bidirectional causal effects, the necessary distinction between confounders and mediators, the possible confounding by weight history, and the possibility of common or upstream underlying causes. In conclusion, causal interpretation of the association is hampered by fundamental conceptual and methodological problems. Experimental studies may elucidate mechanisms, but adequate coverage of the entire pathway from sleep curtailment through obesity development is not feasible. Randomized trials are needed to assess the value of targeted interventions.

Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.

The yield of a diagnostic hospital dyspnoea clinic for the primary health care section.

OBJECTIVE: To investigate the impact of a combined examination programme with treatment advice on patients from general practice with dyspnoea. DESIGN: Prospective study with 6 months followup. SETTING: Regional hospital offering care for patients from 74 general practitioners. SUBJECTS: A total of 284 consecutive patients referred from general practice with dyspnoea. INTERVENTIONS: Patients were subjected to a combined examination programme including physical examination, ECG, chest X-ray, lung spirometry, echocardiography and routine laboratory tests. MAIN OUTCOME MEASURES: (i) Relationship between a diagnosis made by the referring general practitioner and the diagnosis based on the combined examination programme. (ii) The impact of the investigation programme and resulting therapeutic advice on dyspnoea after 6 months. RESULTS: Only in 39% of the patients there was concordance of the diagnoses on referral and the diagnosis based on the examination programme. Heart failure and lung disease was suspected in 126 and 79 patients, respectively, but these diagnoses were confirmed in only one-third to half of the patients. Conversely heart failure was revealed in 13 of 107 patients not suspected of heart failure (12%) and lung disease in 45 of 154 patients not suspected of pulmonary disease (29%). A change of treatment was suggested in 64% of all patients. After 6 months, improvement of dyspnoea was seen in more than half of the patients. In patients in whom the changes of medical treatment were completed, 61% expressed improvement in dyspnoea, whereas improvement of dyspnoea was recorded in only 34% of patients in whom the recommended treatment advice was not taken (P < 0.01). CONCLUSION: (i) In most patients it seems to be too difficult to establish the background of dyspnoea in general practice. (ii) There appears to be a substantial chance of improvement in patients with dyspnoea, in particular for patients who act on treatment advice based on an integrated examination programme; the chance of improvement is almost twice as good as in patients who are not capable to do so.

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor.

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.

Factor VIIa-induced p44/42 mitogen-activated protein kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain.

Signal transduction induced by activated factor VII (FVIIa) was studied with baby hamster kidney (BHK) cells transfected with human tissue factor (TF). FVIIa induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in cells expressing TF, BHK(+TF), but not in wild-type BHK(-TF) cells. BHK(+TF) cells responded to FVIIa in a dose-dependent manner, with detectable phosphorylation above 10-20 nM FVIIa. BHK cells transfected with a cytoplasmic domain-deleted version of TF, (des248-263)TF, or a C245S substitution variant of TF also supported FVIIa-induced MAPK activation. Experiments with active site-inhibited FVIIa, thrombin, factor Xa, and hirudin confirmed that the catalytic activity of FVIIa was mandatory for p44/42 MAPK activation. Furthermore, a high concentration of FVIIa in complex with soluble TF induced p44/42 MAPK phosphorylation in BHK(-TF) cells. These data suggest that TF was not directly involved in FVIIa-induced p44/42 MAPK phosphorylation but rather served to localize the action of FVIIa to the cell surface, potentially to cleave a cell surface receptor. Desensitization experiments with sequential addition of proteases suggested that the p44/42 MAPK response induced by FVIIa was distinctly different from the thrombin response, possibly involving a novel member of the protease-activated receptor family.

Ca2+ in the first epidermal growth factor-like domain of activated factor VII.

Activated factor VII (FVIIa) needs to be bound to tissue factor (TF) to express biological activity, and biochemical and structural data show that the first epidermal growth factor (EGF)-like domain of FVIIa contributes essential contacts with TF. To investigate the role of Ca2+ binding to this domain in FVIIa we used site-directed mutagenesis to replace Asp46 and Asp63 by Asn, which abolished Ca2+ binding, and characterized the double mutant (D46,63N-FVIIa) with respect to its TF-binding properties and the functional status of its complex with TF. D46,63N-FVIIa had a lower amidolytic activity than FVIIa at optimal Ca2+ concentrations. A slightly lower amidolytic activity was also observed in complex with soluble TF, apparently due to a lower catalytic turnover rate of D46,63N-FVIIa. However, D46,63N-FVIIa and FVIIa bound to lipidated TF were equally efficient activators of factor X. The dissociation constant for the interaction between D46,63N-FVIIa and soluble TF, derived from amidolytic activity and direct binding measurements, was approximately 20-fold higher than that for the interaction between FVIIa and soluble TF. The same difference was observed in the affinity for lipidated TF. These findings suggest that a functional Ca2+-binding site in the first EGF-like domain adds approximately 7 kJ/mol to the total binding energy of the interaction with both lipidated and soluble TF.

Bioadhesive drug delivery systems. I. Characterisation of mucoadhesive properties of systems based on glyceryl mono-oleate and glyceryl monolinoleate.

A group of fatty acid esters capable of forming liquid crystals has been identified as a new class of potential bioadhesive substances. The liquid crystals may act as a controlled release system. The experimental work was focused on the monoglycerides, glyceryl mono-oleate (GMO) and glyceryl monolinoleate (GML). The mucoadhesive properties of GMO and GML were demonstrated in vitro by a 'flushing' bioadhesion test system and a tensiometric method. The flushing system was validated with GMO. Mucoadhesion is influenced by the drug and excipient added, their concentrations, and the ability to form especially the cubic phase. It has been shown that the cubic phase is mucoadhesive when formed on wet mucosa, such as rabbit jejunum, and that drug added to the precursor formulation is incorporated in the cubic phase formed. Tensiometric measurements have shown that the unswollen monoglycerides have the greatest mucoadhesion, followed by the partly swollen lamellar phase and the fully swollen cubic phase. The values found for the work of adhesion were in the range 0.007-0.048 mJcm-2. The mechanism of mucoadhesion is unspecific and probably involves dehydration of the mucosa. The cubic phase of GMO and GML may be an interesting candidate for a bioadhesive drug delivery system.

[Artifical respiration with patient in prone position]. / Respiratorbehandling med patienten lejret i bugleje.

The use of mechanical ventilation in prone position was proposed 20 years ago. Since then several investigations have been made trying to explain the mechanism whereby oxygenation is improved when the patient with ARDS is turned into the prone position. In supine position the lung perfusion is highest in the dorsal regions in normal healthy persons. However, when patients with ARDS are turned prone, the predominant dorsal perfusion is partly preserved, while at the same time the dorsal atelectases partly resolve, thereby improving the ventilation/perfusion ratio. Sixty-five percent of patients with early ARDS will achieve significant improvement in oxygenation in prone position. It is recommended that patients with early ARDS that remain hypoxic on mechanical ventilation with PEEP and inspiratory oxygen concentration above 60% be turned prone for 3-6 hours. If effective, the treatment can be repeated once or twice daily until regression of hypoxia. The possible effects of the prone position in other types of acute lung failure are so far not known.

[Patients with acute respiratory distress syndrome mechanically ventilated in prone position]. / Patienter med akut respiratorisk distress-syndrom lejret i bugleje under respiratorbehandling.

Animal experiments and human studies have shown better oxygenation in mechanically ventilated patients with ARDS when the patient is situated in the prone position. In contradiction to former theories of a gravitational gradient of lung perfusion, a number of investigators have found that lung perfusion is preferentially distributed to the dorsal lung regions regardless of body position. The basal atelectasis and oedema in ARDS are resolved and only partly distributed anteriorly in the prone position, and these areas are thereby better ventilated. The combination of better ventilation and unchanged perfusion improves the ventilation/perfusion ratio and decreases the shunt in the prone position. In two cases of prone position in mechanically ventilated patients the PaO2/FiO2 ratio increased from 7.5 to 14.3 and from 8.8 to 19.8 after one hour in the prone position, and some of the improvement was permanent. Prone position has only minor side effects and is recommended as the first choice amongst adjunct therapies in mechanical ventilation in patients with ARDS remaining hypoxic in conventional therapy in the supine position.

Elimination of the Cys558-Cys566 bond in Lys78-plasminogen--effect on activation and fibrin interaction.

Plasminogen contains a unique disulphide bond, Cys558-Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558-Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558-Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.

CD19-selected B lymphocytes synthesize, secrete and migrate in the presence of IL-8. TNF-alpha and gammaIP-10 are also B lymphocyte migratory factors.

B lymphocytes are responsible for antigen uptake and presentation, as well as antibody production. These reactions require close cell-to-cell contact between B lymphocytes and monocytes. In this study we demonstrate that interleukin 8 (IL-8), gamma-immune protein 10 (gammaIP-10) and tumour necrosis factor alpha (TNF-alpha) all induce a significant chemokinetic response of human B lymphocytes. Among the cytokines tested, rIL-8 was the strongest B lymphocyte migratory factor with a migratory index (MI) of 2.03+/-0.32, (P<0.002), followed by rTNF-alpha (MI=1.89+/-0.17, P<0.001) and rgammaIP-10 (MI=1.63+/-0.17, P<0.001). We did not observe B lymphocyte migration towards rIL-1alpha, rIL-2, rIL-4, rIL-10, interferon gamma (rINF-gamma) or transforming growth factor beta (rTGF-beta). Furthermore, we report that human B lymphocytes have a constitutive IL-8 mRNA expression and protein secretion in vitro. Resting as well as stimulated B lymphocytes secrete on average 1.5 ng IL-8/ml medium/24 h (2x10(6) B lymphocytes). Our data indicate a possible mechanism by which B lymphocytes make contact with other cells, during immuno-inflammatory processes.

Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity.

Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in gamma-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca2+ binding site, the function of which is largely unknown. Site-directed mutagenesis of two Ca2+-liganding Asp residues in this domain abolished Ca2+ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca2+ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower kcat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50-60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca2+ binding site in the first epidermal growth factor-like domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.

Site-directed mutagenesis but not gamma-carboxylation of Glu-35 in factor VIIa affects the association with tissue factor.

Factor VIIa is a vitamin K-dependent enzyme whose gamma-carboxyglutamic acid (Gla)-containing domain is important for calcium ion-dependent binding to the cofactor tissue factor and membrane surfaces. This domain contains 10 Gla residues, the individual roles and importance of which are not known. Comparisons with the homologous protein C, factor IX and prothrombin may provide functional information on the first nine Gla residues, whereas no data can be extrapolated to Gla-35 in factor VIIa. Therefore, the effects of posttranslational gamma-carboxylation and site-directed mutagenesis of Glu-35 were investigated. Mutations to Asp, Gln or Val all lead to a lower affinity for tissue factor by decreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Glu or Gla side chains at position 35 appear to fulfil the functional roles equally well.

Prospective 5- to 6-year follow-up study of a cementless acetabular cup.

Twenty-seven patients in whom the Ti-Bac acetabular cup (Zimmer, Warsaw, IN) was placed were examined 5 to 6 years after surgery. The cup was inserted line to line, after reaming without further fixation. In all operations, a cemented Müller straight-stem Protazul femur stem (Zimmer) was used. The patients had good pain relief and improved mobility after the operation. Radiographically, only one hip showed radiolucency in the bone-metal interface after 5 to 6 years. One patient was reoperated 3 days after surgery because of dislocation of the acetabular cup. Apart from this, there were no signs of aseptic loosening of any of these uncemented cups.

[Pulmonary edema after upper airway obstruction]. / Lungeødem efter øvre luftvejsobstruktion.

Post obstructive pulmonary edema (POPE) is a rare, but potentially dangerous condition. We present two patients with post-anaesthetic POPE. The literature is reviewed and aetiology, risk factors, pathogenesis, symptoms, prophylaxis and management are discussed. The condition is often associated with upper airway obstruction related to anaesthesia, but is also related to other causes of upper airway obstruction. Development of pulmonary edema can be delayed for up to 90 minutes. The treatment consists of oxygen therapy by nasal catheter or by mask with continuous positive airway pressure. In severe cases, intubation and mechanical ventilation by respirator with positive end-expiratory pressure is necessary. Further therapy is controversial and without significant effect. With sufficient therapy, almost all patients regain their habitual condition within 24-48 hours and present a normal chest X-ray.

Measurement of neutrophil and eosinophil adhesion to E-selectin, VCAM-1, and ICAM-1 by the use of transfected fibroblast cell lines.

A method which enables the specific measurement of neutrophil and eosinophil adhesion to the endothelial cell adherence receptors E-selectin, VCAM-1 and ICAM-1 has been developed. The method is based on continuous cultures of cell lines of transfected hamster kidney fibroblasts (BHK-21), that selectively express each of the endothelial cell adhesions molecules. Isolated granulocytes are added to the cultured adherent fibroblasts at a ratio of 20:1 and the cells are coincubated for 60 min at 37 degrees C. After removal of the nonadherent granulocytes the amount of adherent granulocytes could be measured by addition of detergent and a peroxidase substrate. Selective measurement of neutrophil and eosinophil adhesion was accomplished by addition of detergent to the adherent cells, collection of extracts followed by measurement of the concentration of an eosinophil (eosinophil cationic protein) and a neutrophil (myeloperoxidase) granule protein, respectively, in the extracts. At basal conditions neutrophils and eosinophils showed significant adhesion to E-selectin and eosinophils a low degree of adhesion to VCAM-1. Significant adhesion of neutrophils and eosinophils to ICAM-1 and of eosinophils to VCAM-1 was selectively induced by addition of manganese ions (Mn2+) at a concentration of 0.5 mmol/l. Neutrophils demonstrated a significantly higher adhesion to E-selectin than eosinophils, while eosinophil adhesion to ICAM-I was significantly higher than that of neutrophils. In conclusion, a method to compare the adhesive capacity of neutrophil and eosinophil granulocytes towards specific endothelial cell adhesion molecules has been developed.

Relationship between genotype, assessed by HLA typing, and phenotypic expression of iron status markers in families of 29 probands with hereditary haemochromatosis.

The purpose of this pedigree study, comprising 29 families with hereditary haemochromatosis (HH), was to evaluate the relationship between the genotype (G), based on HLA typing, and the phenotype, based on measurement of iron status markers (serum transferrin saturation and serum ferritin). Due to tight linkage between the HH locus and the HLA-A locus, 172 relatives of the 29 unrelated probands could be assigned into three groups: G0 who were considered to be normal (n = 53), G1 who were considered to be heterozygotes (n = 105), and G2 who were considered to be homozygotes (n = 14), according to whether they had no, one or two HLA haplotypes in common with the proband. A high serum transferrin saturation (> 60%) was present in 8/14 = 57.1% of the homozygotes, in 11/105 = 10.5% of the heterozygotes, and in 0/53 = 0% of the normals. Of the homozygotes, 8/14 = 57.1% had preclinical disease, 4/14 = 28.6% had clinically overt iron overload, while 2/14 = 14.3% had normal iron status markers. None of the heterozygotes had clinical evidence of iron overload. Analysis of HLA alleles and iron status markers suggested that 11/105 = 10.5% subjects initially classified as heterozygotes (G1) according to HLA typing should be reclassified as homozygotes because of abnormal iron status markers, explained by either: homozygous x heterozygous (n = 7) or heterozygous x heterozygous (n = 2) matings, HLA recombination (n = 1) or strongly abnormal iron status markers (n = 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Prodrugs of thiabendazole with increased water-solubility.

The poor peroral absorption of benzimidazole anthelmintics limits their usefulness for the treatment of systemic infections such as alveolar or cystic echinococcosis. The low bioavailability has mainly been attributed to the low aqueous solubility of the benzimidazoles. Using thiabendazole as a model compound the prodrug approach was investigated as a mean to obtain derivatives with improved water-solubilities. Bioreversible derivatization of thiabendazole was performed by N-acylation of the benzimidazole moiety with various chloroformates as well as by N-acyloxymethylation. Both the N-alkoxycarbonyl and the N-acyloxymethyl derivatives were readily hydrolyzed to thiabendazole in human plasma and in rat and pig liver homogenates. The pH-rate profiles for the hydrolysis of the derivatives were determined and the lipophilicity of the compounds was assessed by partition experiments. The water-solubility of the N-alkoxycarbonyl derivatives was up to 12 times higher than that of the parent drug. An N-(4-amino-methylbenzoyl)oxymethyl derivative possessed a 300-fold higher water-solubility. The improved aqueous solubility, adequate lipophilicity and chemical stability combined with a facile enzymatic hydrolysis make such derivatives promising prodrugs for benzimidazole anthelmintics with the aim of improving the peroral bioavailability.