Expression and Localization of Angiogenic Growth Factors in Developing Porcine Mesonephric Glomeruli

The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations. (J Histochem Cytochem 58:1045–1056, 2010)     

Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid (D/L) ratios exhibit reduced release rates. In this work, the effects of very high D/L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T(1/2)) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T(1/2) = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T(1/2) = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/L ratio increased from 0.025 to 0.1 (wt/wt) (T(1/2) = 6.1-15.6 h respectively) but decreased at higher D/L ratios (D/L = 0.6, wt/wt) (T(1/2) = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.     

Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimination reactions during tandem mass spectrometry

With the emergence of drug resistance and the genomic revolution, there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultralarge enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas-phase carrier domains, facile elimination occurs in benchtop and Fourier-transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This "top down" approach quickly illuminated diverse acyl intermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin, and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2H or 18O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS-PKS projects with benchtop mass spectrometers.     

Intracellular Accumulation of Trehalose Protects Lactococcus lactis from Freeze-Drying Damage and Bile Toxicity and Increases Gastric Acid Resistance

Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.     

Evidence for novel loci for late-onset Parkinson’s disease in a genetic isolate from the Netherlands

We studied patients with idiopathic Parkinson’s disease (PD) from an isolated population in the Netherlands aiming to map gene(s) involved in PD susceptibility. A total of 109 parkinsonism patients were independently ascertained, of whom 62 presented late-onset, idiopathic PD. Genealogical research showed that 45 index cases with idiopathic PD were linked to a common ancestor, indicating familiar clustering among the patients. This strong familial clustering was highly significant (P=0.005) when compared to random controls from the same population. We performed a genome wide scan using 382 polymorphic markers in 44 distantly related PD patients plus 112 unaffected first-degree relatives and spouses. Our genome wide association analysis (DISLAMB) revealed evidence of association at a nominal P-value<0.01 for markers D2S2333, D4S405, D9S158, D13S153. Other regions on chromosomes 3p, 4q, 14q, 17p and 17q were found at a significance level of P<0.05. In a follow-up study, we investigated all the positive regions using a denser marker set and a larger sample (total of 630 individuals including all late-onset PD patients). The strongest evidence for association remained for the 9q and 14q region. A significant association was found for marker D9S1838 (OR=2.0, 95% CI 1.1–3.5, P=0.014) and D14S65 (OR=3.2, 95% CI 1.7–6.1, P<0.001). Moreover, a common haplotype with excess of sharing among late-onset PD cases was observed on both regions. Our results suggest the existence of two loci influencing PD susceptibility on chromosome 9q and 14q.     

Feasibility study on automated recognition of allergenic pollen: grass, birch and mugwort

Quantification of airborne pollen is an important tool in scientific research and patient care in allergy. The currently available method relies on microscopic examination of pollen slides, performed by qualified researchers. Although highly reliable, the method is labor intensive and requires extensive training of the researchers involved. In an approach to develop alternative detection methods, we performed a feasibility study on the automated recognition of the allergenic relevant pollen, grass, birch, and mugwort, by utilizing digital image analysis and pattern recognition tools. Of a total of 254 pollen samples (including 79 of grass, 79 of birch and 96 of mugwort), 97.2% were recognized correctly. This encouraging result provides a promising prospect for future developments.