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AIMS: The aim of this study was to analyse the effect of altered viewing perspectives on the measurement of the glenopolar angle (GPA) and the differences between these measurements made on 3D CT reconstructions and anteroposterior (AP) scapular view radiographs. MATERIALS AND METHODS: The influence of the viewing perspective on the GPA was assessed, as were the differences in the measurements of the GPA between 3D CT reconstructions and AP scapular view radiographs in 68 cadaveric scapulae. RESULTS: The median GPA in 3D reconstructions and AP scapular views were 42.7° (95% confidence intervals (CI), 42.0° to 43.5°) and 41.3° (95% CI 40.4° to 42.0°) respectively (p < 0.001). All but five of 20 malpositions demonstrated a significant difference in GPA compared with the respective AP scapular view (p ≤ 0.005). The GPA was most susceptible to malposition in retroversion/anteversion. Inter- and intra-observer reliability for all measurements of the GPA was excellent for 3D CT reconstructions (intraclass correlation (ICC) 0.93 (95% CI 0.87 to 0.96) and 0.94 (95% CI 0.89 to 0.97), respectively) and higher than on AP scapular radiographs (p < 0.001). The intra- and inter-observer reliability was excellent in AP scapular views and malpositions in extension/flexion (ICC ≥ 0.84) but tended to decrease with increasing viewing angle in retroversion/anteversion. CONCLUSION: These data suggest that 3D reconstructions are more reproducible than AP scapular radiographs in the assessment of the GPA and should be used to compare data in different studies, to predict outcome, define malunion, and act as an indication for surgery in patients with a scapular fracture. Cite this article: Bone Joint J 2016;98-B:1510-16.

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The aim of this study was to identify the incidence of post-operative symptomatic deep-vein thrombosis (DVT), as well as the risk factors for and location of DVT, in 665 patients (701 ankles) who underwent primary total ankle replacement. All patients received low-molecular-weight heparin prophylaxis. A total of 26 patients (3.9%, 26 ankles) had a symptomatic DVT, diagnosed by experienced radiologists using colour Doppler ultrasound. Most thrombi (22 patients, 84.6%) were localised distally in the operated limb. Using a logistic multiple regression model we identified obesity, a previous venous thromboembolic event and the absence of full post-operative weight-bearing as independent risk factors for developing a symptomatic DVT. The incidence of symptomatic DVT after total ankle replacement and use of low-molecular-weight heparin is comparable with that in patients undergoing total knee or hip replacement.

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The topics of verification and validation have increasingly been discussed in the field of computational biomechanics, and many recent articles have applied these concepts in an attempt to build credibility for models of complex biological systems. Verification and validation are evolving techniques that, if used improperly, can lead to false conclusions about a system under study. In basic science, these erroneous conclusions may lead to failure of a subsequent hypothesis, but they can have more profound effects if the model is designed to predict patient outcomes. While several authors have reviewed verification and validation as they pertain to traditional solid and fluid mechanics, it is the intent of this paper to present them in the context of computational biomechanics. Specifically, the task of model validation will be discussed, with a focus on current techniques. It is hoped that this review will encourage investigators to engage and adopt the verification and validation process in an effort to increase peer acceptance of computational biomechanics models.

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The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)-(8alpha)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy-D-nicotine oxidase (6HDNO) by replacing the FAD-binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8-(methylsulfonyl)FAD, and less efficiently with 8-chloro-FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8-(N-acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild-type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.

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Interaction of preproteins with the heat shock protein Hsp70 in the mitochondrial matrix is required for driving protein transport across the mitochondrial inner membrane. Binding of mt-Hsp70 to the protein Mim44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle. However, the available results on the role played by ATP are controversial. Here we demonstrate that the mt-Hsp70.Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70.Mim44 complex in the presence of potassium ions. The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concentration of sodium ions. In the presence of potassium ions, mt-Hsp70 undergoes a conformational change that is not observed with a mutant Hsp70 defective in binding to Mim44. The mutant Hsp70 is able to bind substrate proteins, differentiating binding to Mim44 from binding to substrate proteins. We conclude that binding of ATP, not hydrolysis, is required to dissociate the mt-Hsp70.Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.

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The effect of aniso-osmotic exposure on the level of inducible cyclooxygenase (Cox-2) and on prostanoid synthesis was studied in cultured rat liver macrophages (Kupffer cells). In lipopolysaccharide (LPS)- or phorbol 12-myristate 13-acetate-stimulated Kupffer cells, hyperosmotic (355 mosmol/l) exposure, due to addition of NaCl or impermeant sugars, markedly increased prostaglandin (PG) E2, D2 and thromboxane B2 synthesis in a time- and osmolarity-dependent manner. Increased prostanoid production was observed about 8 h after exposure to LPS in hyperosmotic medium compared to Kupffer cells treated with LPS under normotonic (305 mosmol/l) conditions. A similar stimulatory effect of hyperosmolarity on PGE2 production was also seen when arachidonate was added exogenously. Hyperosmotic stimulation of PGE2 production was accompanied by a strong induction of Cox-2 mRNA levels and an increase in immunoreactive Cox-2, whereas the levels of immunoreactive phospholipase A2 and cyclooxygenase-1 did not change significantly. Dexamethasone, indomethacin and the selective Cox-2 inhibitor, NS-398, abolished the hypertonicity-induced stimulation of PGE2 formation; dexamethasone also prevented the increase in Cox-2 mRNA and protein. The increase of immunoreactive Cox-2 lasted for about 24 h and was also blocked by actinomycin D or cycloheximide, but not by brefeldin A. Tunicamycin or treatment with endoglucosidase H reduced the molecular mass of hypertonicity-induced Cox-2 by 5 kDa. Tunicamycin treatment also suppressed the hypertonicity-induced stimulation of PGE2 production. The hyperosmolarity/LPS-induced stimulation of prostaglandin formation was partly sensitive to protein kinase C inhibition but was not accompanied by an increase in the cytosolic free Ca2+ concentration. The data suggest that osmolarity may be a critical factor in the regulation of Cox-2 expression and prostanoid production in activated rat liver macrophages.

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A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

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Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74-22-15. Two-colour immunofluorescence reveals that My4 and 74-22-15 antigens are coexpressed on the same cells, and cell sorter-purified My4+ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4+ monocytes amongst all leucocytes is 5.8% with 580 cells/microliters. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four-fold lower level on AM. Treatment of cells with phosphatidylinositol-specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74-22-15. Immunoprecipitation with the My4 antibody from surface labelled pig mononuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide- (LPS)-induced interleukin-6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.

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Interleukin-6 has a variety of biological effects, mainly on the immune system. The regulation of this signal at both the site of production and the site of action is necessary to maintain the organism's homeostasis. In the microenvironment of the hepatic sinusoids, Kupffer cells as resident macrophages are the most potent source of interleukin-6 during inflammation. This cytokine is an important signal to hepatocytes during the early stages of the acute-phase response, leading to the expression of several major plasma proteins. Kupffer cells were found to express interleukin-6 receptor constitutively. Interleukin-6 decreased the level of interleukin-6 receptor mRNA, indicating an autocrine pathway by which Kupffer cells regulate their responsiveness to interleukin-6. Furthermore, lipopolysaccharide, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 beta and phorbol ester induced interleukin-6 production and, at the same time, suppressed the level of interleukin-6 receptor mRNA. The existence of an autocrine loop in rat Kupffer cells may be physiologically relevant, as it would contribute to a regulated interleukin-6 signal chain in the liver. The anti-inflammatory mediators dexamethasone or PGE2 and its second messenger, cyclic AMP, increased interleukin-6 receptor mRNA, whereas prostaglandin D2 or the Ca2+ ionophore, A 23187, were without effect. The changes in interleukin-6 mRNA were paralleled by the number of interleukin-6 receptors present on Kupffer cells as detected by binding of 125I-interleukin-6. These results suggest the existence of control mechanisms involving several soluble mediators that help balance the level of interleukin-6-R mRNA in rat liver macrophages.

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Although the nature of the noxious signal and the anatomical target in alopecia areata (AA) are still unknown, it has been assumed that CD4+ T lymphocytes surrounding and infiltrating the hair bulb might trigger the hair loss. As these T lymphocytes do not promote cytotoxic activity we hypothesize that AA is triggered by cytokines. Topical immunotherapy with diphenylcyclopropenone (DCP) is at present the most effective approach. If it is true that AA results from a distinct cytokine pattern, we can hypothesize that the beneficial effect of DCP should be mediated by locally secreted cytokines during the contact allergy. Using semiquantitative reverse transcription-polymerase chain reaction with RNA extracted from scalp biopsies from patients with AA before and after successful treatment with DCP, and from healthy controls we detected a T-cell response with increased steady state mRNA levels for interferon (IFN)-gamma, interleukin (IL)-1 beta, and IL-2 in untreated AA of the totalis type. After DCP treatment, the IFN-gamma expression was reduced but still above the constitutive level found in controls, whereas mRNA expression of IL-2, IL-8, IL-10, and tumor necrosis factor-alpha was increased. Our results point towards cytokines involved in the pathogenesis in AA. A TH1 type cytokine pattern is present in untreated AA, and this is modified by cytokines secreted during DCP treatment. IL-10 has recently been described as an immunomodulator of the TH1 response and, therefore, we hypothesize that basal keratinocytes or lesional T cells secrete bioactive IL-10 after DCP application, resulting in an inhibitory effect on lesional T lymphocytes. This hypothesis would explain the effectiveness of DCP and implies the theoretical possibility of a response to topical or intralesional application of recombinant IL-10.

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Stimulation of cultured human keratinocytes with interleukin (IL)-1 alpha is known to elicit prostaglandin (PG) E2 release. Ultraviolet (UV) B radiation induces keratinocyte PGE2 and cytokine production. The present study deals with the autocrine roles of UVB-induced, keratinocyte-derived cytokines IL-1 and tumor-necrosis-factor (TNF) alpha and their corresponding receptor molecules for UVB-induced PGE2 release. In vitro exposure of transformed human keratinocytes (KB cells) induced PGE2 production five- to eightfold. This increase was inhibited by 70%, if irradiated cells were cultured in presence of monoclonal antibody (MoAb) M4, which blocks IL-1 effects by binding to the type 1 IL-1 receptor (IL-1R). In contrast, MoAb M22, which blocks the type 2 IL-1R, had no significant effects. Addition of recombinant human TNF alpha to unirradiated KB cells resulted in five- to eightfold increased PGE2 synthesis, and this increase could be mimicked by stimulation of KB cells with MoAb htr-9, which exerts TNF alpha-like bioactivity by binding to the 55-kD TNF receptor (TNFR). UVB-induced PGE2 synthesis was blocked by 50% in the presence of neutralizing anti-TNF alpha-Ab, and was completely inhibited by addition of both anti-TNF alpha-Ab and MoAb M4. To elucidate a possible regulatory intracellular step in PGE2 synthesis, specific cyclooxygenase activity in KB cells was determined. Following UVB treatment, cyclooxygenase activity increased twofold, but remained unaltered, if irradiated KB cells were cultured in the presence of anti-TNF alpha-Ab plus MoAb M4. These studies indicate that keratinocyte-derived TNF alpha and IL-1 together mediate UVB-induced PGE2 release via specific cell surface receptors, and that one intracellular mechanism is an increased prostanoid-synthesizing capacity of irradiated cells.

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A novel and reliable high-performance liquid chromatography (HPLC) method is described for the purification and quantification of double-stranded DNA. The nucleic acids may be obtained by polymerase chain reaction (PCR) or as restriction fragments from enzymatic cleavage; the separated products are devoid of contaminating material like agarose, ethidium bromide or non-specific DNA sequences. Because of the non-destructive nature of this HPLC procedure, the purified DNA is optimally suited for cloning experiments. The DNA separation by HPLC has major advantages when combined with reverse transcription (RT)-PCR. This is exemplified by analysis of the TNF-alpha mRNA obtained from endotoxin-elicited rat liver macrophages. If the standard procedure of Northern blotting is compared with the combination of RT-PCR and quantification of the PCR products by HPLC, it is obvious that the dynamic changes of tumor necrosis factor (TNF)-alpha mRNA synthesis are at least as precisely reflected with the RT-PCR/HPLC combination. The latter method is presented as a reliable and powerful tool for quantitative studies on gene expression.

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Prostaglandin-synthesizing activities were demonstrated in cell-free extracts of rat Kupffer cells and characterized. The enzymatic properties of PGH2 synthase were found to be similar to those of synthases present in other organs or cell types. The specific activity of the enzyme was not changed by substances that stimulate prostanoid release by intact Kupffer cells; however, it was reduced by pretreatment of the cells with glucocorticoid hormones. On the other hand, the activities of PGD2 and PGE2 synthase were influenced differently by the kind of cell stimulation. While pretreatment of the intact cells with endotoxin and/or inhibition of protein kinase C led to an enhanced PGE2 formation in cell-free extracts, exposure to agents that enhance protein kinase C-dependent signalling pathways, e.g. phagocytotic stimuli or phorbol ester, suppressed PGE2 synthase activity and, therefore, led to enhanced PGD2 synthesis. It is in line with this observation that in vitro activation of protein kinase C of Kupffer cells resulted in a reduced PGE2 and an enhanced PGD2 synthase activity.

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Sinusoidal endothelial cells were isolated by collagenase-pronase digestion of rat livers followed by centrifugal elutriation. The main endothelial cell fraction consisted of more than 85% endothelial cells as shown by electron microscopy and enzyme histochemistry. Contamination by Kupffer cells was less than 5%. The endothelial cells formed a coherent stable monolayer on dishes coated with collagen type IV in the presence of an RPMI 1640 medium supplemented with 4% Ultroser. Fc receptors were undetectable immediately after elutriation but reappeared after 12 h in culture. Von Willebrand factor (formerly factor VIII-related antigen) could not be detected unequivocally by immunofluorescence. Unchallenged endothelial cells did not produce eicosanoids. In the presence of free arachidonate, however, prostaglandins D2 and E2 as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha were detected by radioimmunoassay and by high-performance liquid chromatography analysis of [3H]arachidonate-exposed cells. Cells treated with the Ca2+ ionophore A23187 produced the same spectrum of immunologically measured prostanoids. In contrast to Kupffer cells in primary culture, eicosanoid formation by endothelial cells was neither triggered by phagocytotic stimuli nor suppressed by pretreatment with dexamethasone.

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The potential of hepatocytes in primary cultures to degrade the prostanoids produced by Kupffer cells and to synthesize eicosanoids, especially leukotriene B4, after treatment with D-galactosamine was studied. Hepatocytes in primary cultures showed a substantial capability to degrade all the prostanoids produced by stimulated Kupffer cells. The rate of degradation, approx. 2 pmol/min per 10(6) hepatocytes, was nearly the same for the prostaglandins D2, E2 and F2a. Lower rates were determined for thromboxane B2 (0.4 pmol/min per 10(6) cells) and for 6-ketoprostaglandin F1a (0.2 pmol/min per 10(6) cells). The degradation products of these prostanoids lacked biological activity, e.g., reactivity with specific antibodies and the ability to contract segments of rabbit femoral artery. In the presence of 30 microM arachidonic acid, hepatocytes produced only very small amounts of prostaglandins and thromboxane, ranging from less than or equal to 22 to 50 fmol/30 min per 10(6) cells. Neither untreated nor D-galactosamine-treated hepatocytes released significant amounts of leukotriene B4. Hepatocytes appear to be the site of degradation rather than synthesis of eicosanoids in the liver.

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The nitrate and nitrite concentration of 96 fasting gastric juice samples of young healthy volunteers were analysed. Two different methods have been used to take the gastric juice: 54 secrete samples were taken directly from the stomach, 42 secrete samples were taken after instillation of 500 ml sterile nitrite and nitrate free NaCl solution ("gastric-washing"). It could be shown that the expected high dilution of nitrite and nitrate concentration after gastric washing does not occur. A possible explanation might be that the solubilisation we could formerly observe in a biological pattern leads to a release of ions. This fact is very important for the question of endogen synthesis of cancerogenic N-nitroso compounds, because high nitrite and nitrate concentrations are subjects of interests as precursors of these compounds.

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The nitrate and nitrite concentrations of 42 fasting gastric juice samples of young healthy human volunteers were analysed. It could be shown by separate determination of the concentration in the real secrete and the mucous gel that the amount of nitrate in the gel is about ten times higher than in the real secrete. The former conception, after which nitrate only gets by swallowed saliva into the stomach, has to be completed. It is obvious that the mucigenous glands of the stomach, like the saliva glands, are also able of nitrate enrichment and secretion. These results are very important for the evaluation of nitrate as a precursor of cancerogenic N-nitroso compounds.

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1. The metabolism of protein and phospholipid in rat liver plasma membranes isolated by the method of Neville [(1960) J. Biophys. Biochem. Cytol. 8, 413-422] was investigated 3 and 6 h after the injection of D-galactosamine in vivo. During this time, all the biochemical and morphological alterations associated with hepatitis developed. 2. After the injection of D-galactosamine the concentration of sphingomyelin in the plasma membrane decreased to below 60% of the control values. 3. The activity of 5'-nucleotidase (EC 3.1.3.5), which has been purified as a sphingomyelin-protein complex, decreased in the total homogenate as well as in the plasma-membrane fraction of livers of rats treated with galactosamine, to about 60% of the control values. 4. Protein synthesis, as measured by the incorporation of [14C]leucine into plasma membranes, was decreased to 45% of that of the controls. However, only small differences were observed in the amino acid composition of the plasma membrane after D-galactosamine treatment. 5. The protein composition of the plasma membranes was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results showed a change from low- to high-molecular-weight proteins after the injection of galactosamine. 6. These results demonstrate different metabolic processes of the plasma membrane altered during the induction of galactosamine hepatitis.

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