Lipid quantitation in formalin-fixed liver sections

We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.     

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.     

Adenylate cyclase activity of membrane fractions isolated from sea urchin sperm

The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I¹²⁵-labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15-fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg⁺⁺ and Na⁺ stimulated their cyclase activity about sevenfold at 2.5 mM Mn⁺⁺ and 3.2 mM ATP. At this ATP to Mn⁺⁺ ratio, high concentrations of Ca⁺⁺ have a small stimulatory effect.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Octanol-water partition coefficient of glucose, sucrose, and trehalose

The octanol-water partition coefficients (P) of glucose, sucrose, and trehalose were measured at temperatures between 5 and 20 degrees C using an enzymatic method. The measured log P is compared with calculated and experimental data previously reported. In the case of trehalose, the measured log P differs considerably from the theoretically estimated values and agrees with the expected value for a disaccharide. Some methods of assessing the partition coefficients are also analyzed and it is concluded that the atom/fragment contribution method overestimates the hydrophilicity of disaccharides and, probably in a larger extension, that of trisaccharides. The knowledge of P for these sugars is valuable both for basic or applied purposes, including food and biomolecules stabilization.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.