Recognition and clearance of methoxypoly(ethyleneglycol)2000-grafted liposomes by macrophages with enhanced phagocytic capacity. Implications in experimental and clinical oncology.

Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two (99m)Tc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). (99m)Tc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (approximately 95%) and yielded products with a high specific activity (approximately 700 Ci/mmol) and good stability (approximately 5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 degrees C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized approximately 3% of added (99m)Tc-sCCK8 per confluent well during 2 h at 37 degrees C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize (99m)Tc-nsCCK8. Displacement of (99m)Tc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 degrees C to prevent internalization) revealed 50% inhibitory concentrations (IC(50)) of 8 nM and >1 microM, respectively. CHO-CCK2 cells internalized approximately 25% and approximately 5% of added (99m)Tc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC(50) values for the displacement of (99m)Tc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected (99m)Tc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of (99m)Tc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of (99m)Tc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of (99m)Tc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of (99m)Tc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with (99m)Tc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate (99m)Tc-nsCCK8, CCK2 tumors accumulate 2 times more (99m)Tc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more (99m)Tc-sCCK8 than (99m)Tc-nsCCK8. Although accumulation in some nontarget organs was also higher with (99m)Tc-sCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of (99m)Tc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.