Efficient autoproteolytic processing of the MHV-A59 3C-like proteinase from the flanking hydrophobic domains requires membranes

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates. In vitro expression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance the trans processing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed from Escherichia coli. Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pelletes. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.     

Synthesis,characterization and properties of photoresponsive polymers comprising photocrosslinkable pendant chalcone moieties

New methacrylate monomers, namely 4‐methacryloyloxyphenyl‐4′‐fluorostyryl ketone and 4‐methacryloyloxyphenyl‐4′‐ethylstyryl ketone comprising a free radical polymerizable group and a photocrosslinkable group, were synthesized by reacting the respective hydroxychalcones with methacryloyl chloride in the presence of triethylamine. The monomers were polymerized in the presence of ethyl methyl ketone (EMK) at 70 °C using benzoyl peroxide as the initiator. The chemical structures were characterized using various spectroscopic techniques: ultraviolet, Fourier transform infrared, ¹H NMR and ¹³C NMR. The thermal stability of the polymers was studied using thermogravimetric analysis in nitrogen atmosphere. Differential scanning calorimetry was used to determine the glass transition temperature of the homopolymers. Photocrosslinking of the synthesized homopolymers was investigated in solution. The two homopolymers were crosslinked within 10–15 min. After crosslinking, the homopolymers were insoluble in the same solvent. Copyright © 2006 Society of Chemical Industry     

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.     

A number-needed-to-treat analysis of the use of respiratory syncytial virus immune globulin to prevent hospitalization

OBJECTIVES: To estimate how many infants in selected high-risk subgroups would require treatment with respiratory syncytial virus immune globulin (RSV-IG) to avoid 1 hospital admission and to determine whether this is economically justified. DESIGN: Cost-benefit analysis. Data from 3 randomized controlled trials of RSV-IG are used to estimate the number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat is computed according to a formula incorporating costs and benefits of RSV-IG prophylaxis. Estimates of the willingness to pay were obtained from a sample of 39 health care providers (35 physicians and 4 nurses). MAIN OUTCOME MEASURES: The number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat that would balance costs with benefits. RESULTS: More than 16 (95% confidence interval, 12.5-23.8) infants would need to be treated with RSV-IG to avoid 1 hospital admission for respiratory syncytial virus infection, ranging from 63 for premature infants without chronic lung disease to 12 (confidence interval, 6.3-100.0) for infants with bronchopulmonary dysplasia. A sensitivity analysis of the costs and values of hospital admission for respiratory syncytial virus infection and RSV-IG treatment resulted in a weak recommendation against the treatment of infants with bronchopulmonary dysplasia and strong recommendations that the costs and risks of RSV-IG treatment outweigh the benefits for the combined sample of infants and premature infants without lung disease. CONCLUSIONS: The number-needed-to-treat procedures offer a method to assess evidence of treatment effects and decision rules for whether to accept treatment recommendations. Under plausible assumptions, treatment with RSV-IG is not recommended for infants without lung disease. Institutions can examine cost and benefit assumptions that best fit their own practice setting.     

Sodium, potassium-activated adenosine triphosphatase activity is impaired in the guinea pig pancreatic duct system in streptozotocin-induced diabetes

In patients with type I diabetes mellitus, clinical studies have demonstrated decreased secretion of pancreatic juice by the pancreatic excretory duct system. The cause of this decrease is unknown, but could involve changes in initial signal transduction pathways or one or more of the electrolyte transport components that subserve regulated fluid secretion. We have compared responsiveness to secretin in pancreatic ducts isolated from healthy and diabetic Hartley guinea pigs and also have compared the expression of CFTR and Na+, K(+)-ATPase in these two groups, as the activities of these two proteins are essential for secretion of pancreatic juice. The increases in cyclic AMP levels evoked by exposure to either 0.1 nM or 0.1 microM secretin were not significantly different in pancreatic ducts isolated from healthy and diabetic guinea pigs nor were levels of CFTR or Na+, K(+)-ATPase expression. By contrast, Na+, K(+)-ATPase activity in pancreatic ducts isolated from diabetic guinea pigs was decreased by 70%, suggesting a change in the enzyme's catalytic properties in the diabetic tissues. The observed decrease would be expected to seriously compromise the production of pancreatic juice.     

Aurally and visually guided visual search in a virtual environment

BACKGROUND: Atopic dermatitis (AD) is characterized by skin infiltrates of leukocytes, such as lymphocytes and eosinophils. OBJECTIVE: To describe the mechanisms determining this inflammatory process, we have analyzed expression of adhesion molecules and their regulation on skin endothelial cells (ECs). METHODS: Expression of adhesion molecules on ECs was analyzed by immunohistochemistry by using Ulex europaeus agglutin 1 as a pan-endothelial marker. RESULTS: Vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin were not found in skin of nonatopic individuals, whereas expression of these surface molecules was observed in nonlesional skin of patients with AD and was even more pronounced in lesional skin or after epicutaneous application of aeroallergen. Induction of adhesion molecule expression was examined on both macrovascular ECs from human umbilical cord vein (HUVECs) and human microvascular ECs (HMEC-1) from skin. TNF-alpha very potently upregulated adhesion molecule expression in vitro on both EC cell types. To verify the in vivo relevance of TNF-alpha, we performed TNF-alpha staining in the skin. TNF-alpha was observed in the dermis of nonatopic skin, both in chymase-containing mast cells and CD68+ macrophages. The increase in the number of TNF-alpha-containing cells was concomitant with the increase in adhesion molecule expression in the skin of patients with AD. IL-4 is supposed to be important in atopic diseases because of its IgE- and VCAM-1-inducing properties. However, IL-4 addition failed to induce VCAM-1 expression on HMEC-1, although in the same set of experiments, a clear induction of VCAM-1 expression by IL-4 on HUVECs was demonstrated. Flow cytometry revealed the absence of 11-4 receptor alpha-chains on HMEC-1 and their presence on HUVECs. Immunohistochemistry examination on skin sections showed no binding of the IL-4R alpha-chain antibodies to ECs. CONCLUSION: We conclude that adhesion molecule expression is increased in the skin of patients with AD. Most probably, this increased expression is not a (direct) effect of IL-4 on skin endothelium, but other cytokines, such as TNF-alpha, might be responsible for this increased adhesion molecule expression. Continuous adhesion molecule expression may facilitate T-cell extravasation in a nonantigen-specific manner, thus explaining the presence of increased T-cell numbers in nonlesional skin of patients with AD.     

The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.