Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.     

Nebulisers or spacers for the administration of bronchodilators to those with asthma attending emergency departments?

BACKGROUND: Systematic reviews and national guidelines conclude that the nebulised route of administration of bronchodilators has no advantage over the use of a spacer in moderately severe exacerbations of asthma. Whether this recommendation is implemented and whether it might affect use of staff time is unknown. OBJECTIVES: To determine the current method of administration of bronchodilators to those with non-life-threatening asthma attending emergency departments (ED) in London, UK and to monitor the implementation of a new policy to administer bronchodilators by spacers in one ED with a special reference to the time taken by nurses to administer the therapy by two different routes. METHODS: Thirty-five EDs in Greater London were surveyed regarding their current practice. A time and motion study was then undertaken in one department observing nurses administering bronchodilators in the 3 weeks before and 3 weeks after a departmental policy change to favour the use of spacer devices rather than nebulisers. RESULTS: The majority of EDs (94.3%) in Greater London were using the nebulised route of administering bronchodilators to the majority of their adult patients. Spacers were more commonly used for the treatment of children (60.3% of departments using spacers and nebulisers or spacers alone). Over half of the hospitals surveyed (51.4%) were unaware that the British Guidelines on Asthma Management suggested that outcomes were the same and that there were potential advantages in the use of a spacer for both adults and children. Time and motion studies showed that the use of a spacer took no more nursing time than administration of the bronchodilator via a nebuliser; in fact treatment and set-up time were considerably lower for spacers. CONCLUSION: Spacer administration of bronchodilators to those with asthma attending EDs utilises less treatment time than use of a nebuliser. A survey of EDs in Greater London has shown that despite guideline conclusions there appears to be little evidence of reduction in use of nebulisers; a fear that use of alternatives might take nurses longer is not supported by this study.     

Insulin resistance is associated with increased serum levels of glycosylphosphatidylinositol-specific phospholipase D

The dyslipidemia of the metabolic syndrome is associated with alterations in triglyceride and high-density lipoprotein (HDL) metabolism. We examined the serum levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a minor HDL-associated protein, in a cohort with a wide range of insulin sensitivity. The mean serum GPI-PLD mass from 109 subjects was 58.9 +/- 18.4 microg/mL (mean +/- SD). GPI-PLD levels directly correlated with cholesterol, apolipoprotein AI, triglycerides, insulin, and homeostasis model assessment (HOMA) but not C-reactive protein. These results suggest that increased serum GPI-PLD is associated with the insulin resistance.     

The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis

The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30 kDa) is much faster (t(1/2) = 4-5 h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane potential (-delta psi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.     

Case Report: Adrenal Oncocytoma Associated with Markedly Increased FDG Uptake and Immunohistochemically Positive for GLUT1

Usually, benign tumors are not associated with an increased F-18 fluorodeoxyglucose (F-18 FDG) uptake on positron emission tomography (PET), although some exceptions have been reported in adrenal neoplasms. We present a rare case of adrenocortical oncocytoma associated with markedly increased FDG uptake, demonstrating a maximum standardized uptake value of 46.8. Histological examination demonstrated diffuse proliferation of tumor cells with eosinophilic and granular cytoplasm that were diffusely immunopositive for mitochondria and glucose transport protein 1, with focal and weak immunopositivity for 3β-hydroxysteroid dehydrogenase. Ultrastructural examination also revealed abundant mitochondria in the tumor cells. The tumor was diagnosed as adrenocortical oncocytoma and was considered benign according to Lin-Weiss-Bisceglia criteria. Diagnosis of adrenocortical oncocytoma can pose difficulties during both preoperative radiological and postoperative histopathological investigations.     

Patient reported experiences of CT guided lung biopsy: a prospective cohort study

Background

CT guided lung biopsy is a commonly performed procedure to obtain tissue for a histological diagnosis in cases of suspected lung cancer.

Methods

This is a prospective cohort study to obtain information directly from patients about their experiences of the biopsy procedure, thus obtaining a more accurate picture of complications compared with previously performed retrospective reviews. Patients participated in a post-procedure telephone interview and information was gathered about any procedural complications and personal experiences. We also compared the patient reported complications with those obtained from a retrospective review of hospital databases, analogous to previously performed retrospective studies.

Results

In our patient group, reported procedural complication rates were 10% pneumothorax rate (4% requiring a chest drain) and 10% haemoptysis. Post-procedural pain and shortness of breath showed positive correlation, with one patient experiencing prolonged pain. No statistical difference was found between the patient reported complication rates and those obtained from retrospective review of the hospital database.

Conclusions

Our study demonstrates CT guided lung biopsy is a safe procedure and is generally well tolerated. Some patients may experience significant and lasting pain and therefore should be counselled about this pre-procedure.

     

Target‐ and input‐dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus

We examined the synaptic structure, quantity, and distribution of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐ and N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze‐fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN‐BC synapses) and fusiform cells (AN‐FC synapses) and PF synapses on FC (PF‐FC synapses) and cartwheel cell spines (PF‐CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target‐dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input‐dependent organization in FCs. Among the four excitatory synapse types, the AN‐BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF‐CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP‐cluster area and the AMPAR number, indicating a common intrasynapse‐type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold‐labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP‐cluster area and the NMDAR number. Our observations reveal target‐ and input‐dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses. J. Comp. Neurol. 522:4023–4042, 2014. © 2014 Wiley Periodicals, Inc.