Bovine factor v: a calcium-containing metalloprotein

Although coagulation factor Xa requires Ca2+ for binding to phospholipid, factor V, the other protein component in the prothrombinase enzyme complex, binds tightly to phospholipid in the absence of Ca2+. To explore the possibility that calcium might be present in the fact V molecule, the effect of several chelators, including oxalate, citrate, pyrophosphate, and EDTA, on factor V activity has been studied. A time- and concentration-dependent inhibition of factor V which reflects the respective association constants for calcium of each chelator is observed. The inhibition can be prevented by the prior addition of calcium and manganese but not magnesium. Reversal of the activity loss can be accomplished at high protein concentrations by the addition of calcium, the removal of the chelator by gel filtration, or an increase in temperature. Factor V contains 1 g atom of calcium per 300,000 daltons which is not removed by incubation with EDTA under nondenaturing conditions. Thus, the inhibition by EDTA is due to binding to calcium associated with factor V. In 8 M urea, EDTA can remove over 80% of the calcium, demonstrating the importance of the native structure in maintaining the calcium binding site. Prior binding of phospholipids to factor V prevents inhibition by EDTA. The results suggest that phospholipids complex at the calcium site on the factor V metallopretein.     

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47- Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.     

Immune complexes containing factor V in a patient with an acquired neutralizing antibody

An 82-year-old woman presented with extensive hematomas and melena associated with markedly decreased plasma factor V coagulant activity (FV:C). Using a competitive enzyme-linked immunosorbent assay developed in our laboratory, we made serial measurements of factor V antigen (FV:Ag) in plasma and found it to be normal or elevated. The patient's plasma was demonstrated to contain an IgG antibody that could neutralize FV:C in normal plasma. The antibody was of restricted heterogeneity (IgG1, IgG2,kappa). Circulating immune complexes containing antibody to factor V and FV:Ag were demonstrated directly in the plasma by immunoelectrophoresis with polyclonal monospecific antibody and with a monoclonal antibody using an enzyme-linked immunosorbent assay. Presence of neutralizing antibody could be demonstrated in vitro even at times when FV:C was within normal limits by heat inactivation of FV:C. Treatment with plasma and platelet transfusions as well as plasmapheresis induced definite but transient elevation of FV:C. Steroid therapy lowered the neutralizing antibody concentration and produced a rapid and persistent elevation of FV:C during two separate hospitalizations. This report describes a patient in whom levels of FV:Ag have been serially measured, and the presence of circulating immune complexes consisting of factor V and a neutralizing antibody have been directly demonstrated.     

Crotalocytin: characterization of the timber rattlesnake platelet activating protein

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.     

Purification and functional evaluation of mature neutrophils from human bone marrow

Human myeloid maturation proceeds within the bone marrow and results in a mature neutrophil that is released into the peripheral circulation. Previous reports have indicated that neutrophils from bone marrow demonstrate decreased adherence, impaired phagocytosis, and decreased nitroblue tetrazolium dye reduction when stimulated. Due to lack of a suitable method for isolating purified bone marrow neutrophils, these studies have been performed by microscopic techniques. We now report a method for isolating 1 X 10(8) neutrophils [bands plus polymorphonuclear leukocytes (PMNs)] from 10 mL of bone marrow aspirate sample. By means of a discontinuous Percoll-gradient centrifugation through densities of 1.085, 1.095, and 1.10 g/mL a leukocyte-rich suspension of bone marrow can be separated into three leukocyte layers. By combining the lower two leukocyte layers (M2/3), a population of neutrophils consisting of 26% bands and 63% PMNs is seen. When compared with peripheral blood PMNs, these bone marrow neutrophils had a lower alkaline phosphatase activity, decreased ingestion of Oil Red O-coated particles, impaired superoxide release on stimulation with the chemotactic peptide Fmet-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA), and less activity of the NADPH-dependent oxidase. These results indicate that morphologically mature neutrophilic cells within the bone marrow exist in a still functionally immature state.     

Differential actions of ethanol and trichloroethanol at sites in the M3 and M4 domains of the NMDA receptor GluN2A (NR2A) subunit

Background and purpose:

Alcohol produces its behavioural effects in part due to inhibition of N-methyl-d-aspartate (NMDA) receptors in the CNS. Previous studies have identified amino acid residues in membrane-associated domains 3 (M3) and 4 (M4) of the NMDA receptor that influence ethanol sensitivity. In addition, in other alcohol-sensitive ion channels, sedative-hypnotic agents have in some cases been shown to act at sites distinct from the sites of ethanol action. In this study, we compared the influence of mutations at these sites on sensitivity to ethanol and trichloroethanol, a sedative-hypnotic agent that is a structural analogue of ethanol.

Experimental approach:

We constructed panels of mutants at ethanol-sensitive positions in the GluN2A (NR2A) NMDA receptor subunit and transiently expressed these mutants in human embryonic kidney 293 cells. We used whole-cell patch-clamp recording to assess the actions of ethanol and trichloroethanol in these mutant NMDA receptors.

Key results:

Ethanol sensitivity of mutants at GluN2A(Ala825) was not correlated with any physicochemical measures tested. Trichloroethanol sensitivity was altered in two of three ethanol-insensitive mutant GluN2A subunits: GluN2A(Phe637Trp) in M3 and GluN2A(Ala825Trp) in M4, but not GluN2A(Met823Trp). Trichloroethanol sensitivity decreased with increasing molecular volume at Phe637 or increasing hydrophobicity at Ala825 and was correlated with ethanol sensitivity at both sites.

Conclusions and implications:

Evidence obtained to date is consistent with a role of GluN2A(Ala825) as a modulatory site for ethanol and trichloroethanol sensitivity, but not as a binding site. Trichloroethanol appears to inhibit the NMDA receptor in a manner similar, but not identical to, that of ethanol.     

Conservatively treated massive prolapsed discs: a 7-year follow-up

INTRODUCTION

The natural history of a lumbar hernia of the nucleus pulposus (HNP) is not fully known and clear indications for operative intervention cannot be established from the literature. Several studies have shown that the largest discs appear to have the greatest tendency to resolve. The aim of this study was to investigate whether massive prolapsed discs can be safely managed conservatively once clinical improvement has occurred.

PATIENTS AND METHODS

Thirty-seven patients were studied by clinical assessments and serial magnetic resonance imaging (MRI) over 2 years. Patients had severe sciatica at first, but began to show clinical improvement despite the large disc hernia-tions. Clinical assessment included the Lasegue test and neurological appraisal. The Oswestry Disability Index was used to measure function and changes in function. Serial MRI studies allowed measurement of volume changes of the herniated disc material over a period of time.

RESULTS

Initial follow-up at an average of 23.2 months revealed that 83% had a complete and sustained recovery at the initial follow-up. Only four patients required a discectomy. The average Oswestry disability index improved from 58% to 15%. Volumetric analysis of serial MRI scans found an average reduction of 64% in disc size. There was a poor correlation between clinical improvement and the extent of disc resolution.

CONCLUSIONS

A massive disc herniation can pursue a favourable clinical course. If early progress is shown, the long-term prognosis is very good and even massive disc herniations can be treated conservatively.     

Target setting in intensive insulin management is associated with metabolic control: the Hvidoere Childhood Diabetes Study Group Centre Differences Study 2005

Swift PGF, Skinner TC, de Beaufort CE, Cameron FJ, Åman J, Aanstoot H‐J, Castaño L, Chiarelli F, Daneman D, Danne T, Dorchy H, Hoey H, Kaprio EA, Kaufman F, Kocova M, Mortensen HB, Njølstad PR, Phillip M, Robertson KJ, Schoenle EJ, Urakami T, Vanelli M, Ackermann RW, Skovlund SE for the Hvidoere Study Group on Childhood Diabetes. Target setting in intensive insulin management is associated with metabolic control: the Hvidoere Childhood Diabetes Study Group Centre Differences Study 2005. Objective: To evaluate glycaemic targets set by diabetes teams, their perception by adolescents and parents, and their influence on metabolic control. Methods: Clinical data and questionnaires were completed by adolescents, parents/carers and diabetes teams in 21 international centres. HbA1c was measured centrally. Results: A total of 2062 adolescents completed questionnaires (age 14.4 ± 2.3 yr; diabetes duration 6.1 ± 3.5 yr). Mean HbA 1c = 8.2 ± 1.4% with significant differences between centres (F = 12.3; p < 0.001) range from 7.4 to 9.1%. There was a significant correlation between parent (r = 0.20) and adolescent (r = 0.21) reports of their perceived ideal HbA1c and their actual HbA1c result (p < 0.001), and a stronger association between parents' (r = 0.39) and adolescents' (r = 0.4) reports of the HbA1c they would be happy with and their actual HbA1c result. There were significant differences between centres on parent and adolescent reports of ideal and happy with HbA1c (8.1 < F > 17.4;p < 0.001). A lower target HbA1c and greater consistency between members of teams within centres were associated with lower centre HbA1c (F = 16.0; df = 15; p < 0.001). Conclusions: Clear and consistent setting of glycaemic targets by diabetes teams is strongly associated with HbA1c outcome in adolescents. Target setting appears to play a significant role in explaining the differences in metabolic outcomes between centres.     

Routine microbiological testing in sudden and unexpected infant death

Objective : To evaluate the significance of microbiological test results in a series of infants who had died suddenly and unexpectedly.
Methodology : Following a review of all cases of sudden natural death in infants presenting to the Adelaide Children's Hospital (ACH) division of the Women's and Children's Hospital (WCH) over the 10 year period between 1983 and 1992, specific evaluation of microbiological test results was undertaken.
Results : There were 329 cases of sudden infant death syndrome (SIDS) and 23 cases in which sudden infant death was either attributed to other conditions or was unclassifiable. Positive microbiological results were recorded in the majority of cases, most being considered to be due to postmortem overgrowth or to contamination at autopsy. Of the remaining cases, microbiological results were essential to the establishment of the diagnosis in three cases, and were a useful adjunct to the diagnosis in a further six cases.
Conclusions : Routine microbiological testing in cases presenting as SIDS did not reveal occult sepsis in most instances. Such testing did, however, add support to the diagnosis of SIDS where no pathogens were isolated and, if not undertaken, would have resulted in a small percentage of cases of sudden infant death due to infections remaining undiagnosed.