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Objective: To study genetic variants and their function within genes coding for complement receptors in pre‐eclampsia. Design: A case–control study. Setting: Pre‐eclampsia is a common vascular disease of pregnancy. The clearance of placenta‐derived material is one of the functions of the complement system in pregnancy. Population: We genotyped 500 women with pre‐eclamptic pregnancies and 190 pregnant women without pre‐eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre‐eclamptic pregnancies and 1905 controls from the national FINRISK cohort. Methods: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS‐1 cells. Main outcome measures: Allele frequencies were compared between pre‐eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. Results: The most significantly pre‐eclampsia‐linked CR3 variant M441K (P = 4.27E‐4, OR = 1.401, 95% CI = 1.167–1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. Conclusions: Results suggest that changes in complement‐facilitated phagocytosis are associated with pre‐eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro‐ and anti‐inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre‐eclampsia pathogenesis. Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre‐eclampsia. Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre‐eclampsia. This article includes Author Insights, a video abstract available at https://vimeo.com/bjog/authorinsights16660 [ABSTRACT FROM AUTHOR]

Objective: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia., Design: A case-control study., Setting: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy., Population: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort., Methods: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells., Main Outcome Measures: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays., Results: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b., Conclusions: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis., Tweetable Abstract: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia., (© 2021 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Introduction: Glycogen storage disease V (GSDV, McArdle disease) and GSDVII (Tarui disease) are the most common of the rare disorders of glycogen metabolism. Both are associated with low lactate levels on exercise. Our aim was to find out whether lactate response associated with exercise testing could distinguish between these disorders., Methods: Two siblings with Tarui disease, two patients with McArdle disease and eight healthy controls were tested on spiroergometric exercise tests with follow-up of venous lactate and ammonia., Results: A late increase of lactate about three times the basal level was seen 10-30 min after exercise in patients with Tarui disease being higher than in McArdle disease and lower than in the controls. Ammonia was increased in Tarui disease., Discussion: Our results suggest that follow-up of lactate associated with exercise testing can be utilized in diagnostics to distinguish between different GSD diseases.

Integrins are adhesion molecules on the surface of cells. In blood cells they are responsible for rapid changes during adhesion of the cell to the endothelium. Deficiency or defective function of integrins will result in severe illnesses. Surprisingly, certain variants of integrins are associated with an increased risk of developing SLE. In autoimmune diseases and as a result of organ transplantations integrins participate in reactions in which leukocytes attack the body's own tissues. This has resulted in the development of drugs in antibody form for inhibition of the action of integrins. These drugs may, however, exhibit severe adverse effects.

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.

Background: The triggering agent of multiple sclerosis is still unknown and many viruses, including human herpesvirus-6 (HHV-6), are under suspicion. In earlier study we found patients who had HHV-6 reactive OCBs in their CSF. We wanted to investigate whether HHV-6 has an active role in diseases with demyelination., Objective: To analyze the HHV-6-reactive cases in detail and investigate the possible independent role of HHV-6 in the development of central nervous system involvements with demyelination., Study Design: We studied serum and CSF samples that were collected over a period of one year, from all patients who had oligoclonal bands (OCB) in cerebrospinal fluid (CSF) and were examined in the Department of Neurology, University Central Hospital of Helsinki, Finland. Clinical evaluation was accomplished blinded of HHV-6 analysis and follow-up time was two years. All patients underwent MRI of the head and clinically indicated CSF analysis., Results: The 17 patients with HHV-6-reactive OCBs were significantly younger and had significantly more IgG-OCBs in comparison to patients without HHV-6-reactive OCBs. Initial diagnoses in patients with HHV-6-reactive OCBs remained the same during the follow-up time., Conclusion: Patients with HHV-6-positive OCBs appear to form a separable group. In progressive neurological diseases HHV-6 may have a role in long-term infection with demyelination., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Demyelinating diseases of the central nervous system (CNS) often include elevated IgG production in intrathecal space presenting as oligoclonal bands (OCBs) in cerebrospinal fluid (CSF). In most demyelinating diseases, e.g. in multiple sclerosis (MS), the underlying cause is not known. We used isoelectric focusing and affinity immunoblot to study the specificity of CSF OCBs to human herpesvirus-6 (HHV-6) in patients with demyelinating diseases of the CNS including MS. Eighty patients with positive OCB finding were included in the study. The OCBs reacted with the HHV-6 antigen in 18 cases (23%). Twelve of 46 MS patients (26%), 5 of 24 other demyelinating diseases (21%) and 1 of 10 other neurological disorders (10%) had HHV-6 specific OCBs in CSF. A specific intrathecal HHV-6 A and B antibody production was shown in a proportion of patients with demyelinating diseases and might suggest a role in the pathogenesis of these diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Subarachnoid hemorrhage typically starts abruptly with an intense ("thunderclap") headache but sometimes the symptoms are atypical. The mortality is approximately 50%. Correct immediate diagnosis is important because the hemorrhage is usually caused by a treatable aneurysm. Computer tomography (CT) is the principal diagnostic method on arrival to the emergency room. If the CT is negative and the situation remains unclear, examination of the cerebrospinal fluid is essential and should entail analysis of heme pigments, especially in warning leaks and after delayed admission. Only spectrophotometry of the sample is sensitive enough and should replace visual inspection.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Objective: Creatine kinase and myoglobin are markers of muscular damage in rhabdomyolysis. Whereas myoglobin is considered to be the principal compound causing tubular damage, serum creatine kinase level is presently guiding therapeutic interventions in clinical practice to prevent acute renal failure. Because differences in elimination kinetics of these two compounds may influence therapeutic decisions, we studied elimination kinetics of myoglobin and creatine kinase in patients with rhabdomyolysis., Design: Open, noncomparative study., Setting: Intensive and intermediary care units in a university hospital., Patients: A total of 13 consecutive patients with rhabdomyolysis whose baseline serum creatine kinase exceeded 5000 IU/L. Ten of 13 patients were treated with forced alkaline diuresis, and none were dialyzed., Results: Myoglobin had faster elimination kinetics than creatine kinase (p <.01), and the average times to reach the 50% level of initial values were 12 hrs for myoglobin and 42 hrs for creatine kinase. Elimination of myoglobin was not affected by glomerular filtration rate. Compared with creatinine clearance (mean, 102 mL/min), myoglobin clearance was low (mean, 3 mL/min), both in patients with preserved renal function (n = 11) and in those with acute renal failure (n = 2)., Conclusion: Serum myoglobin has faster elimination kinetics than creatine kinase in patients treated with forced alkaline diuresis for rhabdomyolysis. Considering the etiologic role of myoglobin, our data suggest that serum myoglobin level, rather than that of creatine kinase, should be used to guide therapy in patients with rhabdomyolysis.

We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50 degrees C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae.

Mannose is an aldohexose component of a number of glycoproteins in cellular membranes and blood plasma. Free (unbound) mannose is a normal blood plasma constituent and its concentration is elevated in diabetes mellitus and chronic glomerulonephritis. We devised an enzymatic method for the determination of free mannose in which mannose is converted to glucose-6-phosphate and measured spectrophotometrically using glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide phosphate (NADP). Accumulation of reduced NADP in the assay was verified by spectral analysis and by finding rapid disappearance of absorbance at 340 nm on addition of glutathione reductase and oxidized glutathione into the reaction mixture. The method necessitates prior removal of glucose from the samples. This we accomplished using glucose-6-phosphate dehydrogenase and a surplus amount of NADP, followed by elimination of reduced NADP by acidification of the reaction mixture. The assays may be run in parallel for expediency. Concentration of free mannose in serum was 18.5 +/- 5.5 mumol/l in healthy fasting female adults. The analytical recovery was 90.2 +/- 10.2% and the between-run imprecision was 13.5% (18.5 +/- 5.5 mumol/l, mean +/- SD) and 10.4% (75.3 +/- 10.3 mumol/l). The assay showed rectilinearity up to 220 mumol/l, which covers the measuring range to which the mannose concentrations in normal and clinical samples may be expected to fall.

Vitamin K functions in animal cells as the cofactor of the enzyme vitamin K-dependent carboxylase which catalyzes the post-translational formation of gamma-carboxyglutamyl (Gla) residues in specific vitamin K-dependent proteins. These proteins include four blood coagulation factors (prothrombin and Factors VII, IX and X), other plasma proteins (protein C, protein S and protein Z), two proteins from bone (osteocalcin or bone Gla-protein and matrix Gla-protein), and other proteins from lung, kidney, spleen, testis, placenta and other tissues. In the proteins involved in blood coagulation the Gla residues are mandatory for the activation of the inactive proenzymes; this process occurs on phospholipid surfaces to which the proenzymes are bound via Gla residues and calcium ions. The energy needed in the carboxylation reaction is obtained from the oxidation of vitamin K hydroquinone to 2,3-epoxide of the vitamin. Specific enzymes, vitamin K epoxide reductase and vitamin K quinone reductases, catalyze consecutive reactions in which the vitamin K hydroquinone is regenerated, thus allowing continued use of the vitamin K molecule for the carboxylations. The oral anticoagulants, derivatives of 4-hydroxycoumarin and indan-1,3-dione, used as therapeutic agents in thromboembolic disease, are antagonists to vitamin K preventing the catalytic use of vitamin K in the carboxylations by irreversibly inhibiting vitamin K epoxide reductase.

Red cell hemolysates from a Finnish population sample (n = 242) were fractionated by isoelectric focusing on polyacrylamide gel, and S-formylglutathione hydrolase (EC 3.1.2.12) was located by activity staining. Polymorphism, which is probably genetically determined, was found. The samples from most persons studied gave one major enzyme band, whereas for 6 persons three enzyme bands were found. The enzyme is a dimer, and the polymorphism observed appears to result from two alleles, FGH1 and FGH2 at an autosomal locus. The frequency found for FGH1 was 0.988.

The steady-state kinetic mechanism of vitamin K-dependent carboxylase from calf liver has been investigated by initial-velocity measurements with varying concentrations of two carboxylase substrates and constant, nonsaturating concentrations of the other two substrates. With all combinations of the varied substrates tested linear kinetics were obtained with lines intersecting on the left side of the 1/v axis in double-reciprocal plots. Thus the carboxylase has a sequential reaction mechanism which includes the quinternary complex of the enzyme with its four substrates. A mechanism with the ordered steady-state addition of all substrates to the enzyme accords well with the results. A totally random mechanism was excluded but the alternative possibility remained that part of the substrates are added in a rapid-equilibrium random reaction. Experiments with saturating constant concentrations of sodium bicarbonate and varying concentrations of the other substrates suggest that bicarbonate (CO2) is either the first or, more probably, the last substrate bound to the enzyme.

Methods have been devised for the separation of the isoenzymes of glyoxalase I(S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from human red blood cells by electrophoresis and electrofocusing on polyacrylamide gel slabs. Three different staining methods were used for the location of the enzyme. Three electrophoretic phenotypes of the enzyme were resolved, the fast and slow types with one band and the intermediate type with three glyoxalase I activity bands. In gel electrofocusing (pH gradient 3.5-9.5) two glyoxalase I activity bands were found for all electrophoretic types. In electrofocusing on gel with a narrow pH gradient, at least four separate enzyme components were resolved for the fast and slow electrophoretic types and at least six components for the intermediate type. The phenotypes could be distinguished correspondingly to the electrophoretic results. Preparative separation of the isoenzymes were achieved by ion exchange chromatography on DEAE-Sephacel but gel chromatography on Sephadex G-100 gave the same elution volume for all enzyme phenotypes. This corresponds to an apparent molecular weight of about 47 000.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.