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.     

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate (PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate = 312 s(-1)), PEG-diisocyanate treated surfaces experienced 96% (PE), 97% (PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for 1 h prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen-coated glass coverslips and platelet deposition onto preclotted Dacron were also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface-associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.     

Minimally invasive mitral valve repair using transthoracic aortic occlusion

This report describes a minimally invasive mitral valve repair done through a limited (6-cm) thoracic incision. The patient was supported by peripheral extracorporeal perfusion with cardiac arrest established using a new transthoracic aortic cross-clamp and antegrade blood cardioplegia. The patient was discharged on postoperative day 3 with minimal pain. This less invasive approach to mitral valve surgery may offer combined advantages to patients by increasing comfort, expediting recovery, and decreasing surgical costs by using modified traditional methods compared with specialized intraaortic occlusive balloons.     

Reduction of aerobic acetate production by Escherichia coli

Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.     

Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use

Data generated in the Plasmodium yoelii rodent model indicated that plasmid DNA vaccines encoding the P.yoelii circumsporozoite protein (PyCSP) or 17 kDa hepatocyte erythrocyte protein (PyHEP17) were potent inducers of protective CD8+ T cell responses directed against infected hepatocytes. Immunization with a mixture of these plasmids circumvented the genetic restriction of protective immunity and induced additive protection. A third DNA vaccine encoding the P. yoelii sporozoite surface protein 2 (PySSP2) also induced protection. The P. falciparum genes encoding the homologues of these three protective P. yoelii antigens as well as another P. falciparum gene encoding a protein that is expressed in infected hepatocytes have been chosen for the development of a human vaccine. The optimal plasmid constructs for human use will be selected on the basis of immunogenicity data generated in mice and nonhuman primates. We anticipate that optimization of multi-gene P. falciparum DNA vaccines designed to protect against malaria by inducing CD8+ T cells that target infected hepatocytes will require extensive clinical trials during the coming years.     

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.