Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.     

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.     

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.