Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of HDL cholesteryl esters is well established. In SR-BI-deficient mice, we recently observed a delayed postprandial triglyceride (TG) response, suggesting an additional role for SR-BI in facilitating chylomicron (CM) metabolism. Here, we assessed the effect of adenovirus-mediated hepatic overexpression of SR-BI (Ad.SR-BI) in C57BL/6J mice on serum lipids and CM metabolism. Infection of 5 x 10(8) plaque-forming units per mouse of Ad.SR-BI significantly decreases serum cholesterol (>90%), phospholipids (>90%), and TG levels (50%), accompanied by a 41.4% reduction (P < 0.01) in apolipoprotein B-100 levels. The postprandial TG response is 2-fold lower in mice treated with Ad.SR-BI compared with control mice (area under the curve = 31.4 +/- 2.4 versus 17.7 +/- 3.2; P < 0.05). Hepatic mRNA expression levels of genes known to be involved in serum cholesterol and TG clearance are unchanged and thus could not account for the decreased plasma TG levels and the change in postprandial response. We conclude that overexpression of SR-BI accelerates CM metabolism, possibly by mediating the initial capture of CM remnants by the liver, whereby the subsequent internalization can be exerted by additional receptor systems such as the LDL receptor (LDLr) and LDLr-related protein 1.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.     

Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns

Genome and gene duplications are considered to be the impetus to generate new genes, as the presence of multiple copies of a gene allows for paralogues to adopt novel function. After at least two rounds of genome/gene duplication, the Runt gene family consists of three members in vertebrates, instead of one in invertebrates. One of the family members, Runx2, plays a key role in the development of bone, a tissue that first occurs in vertebrates. The family has thus gained new gene function in the course of evolution. Two Runx2 genes were cloned in the vertebrate model system the zebrafish (Danio rerio). The expression patterns of the two genes differ and their kinetics differ up to four fold. In addition, splice forms exist that are novel when compared with mammals. Together, these findings comprise opportunities for selection and retention of the paralogues towards divergent and possibly new function.     

Pathogen adaptation under imperfect vaccination: implications for pertussis

Mass vaccination campaigns have drastically reduced the burden of infectious diseases. Unfortunately, in recent years several infectious diseases have re-emerged. Pertussis poses a well-known example. Inspired by pertussis, we study, by means of an epidemic model, the population and evolutionary dynamics of a pathogen population under the pressure of vaccination. A distinction is made between infection in immunologically naive individuals (primary infection) and infection in individuals whose immune system has been primed by vaccination or infection (secondary infection). The results show that (i) vaccination with an imperfect vaccine may not succeed in reducing the infection pressure if the transmissibility of secondary infections is higher than that of primary infections; (ii) pathogen strains that are able to evade the immunity induced by vaccination can only spread if escape mutants incur no or only a modest fitness cost and (iii) the direction of evolution depends crucially on the distribution of the different types of susceptibles in the population. We discuss the implications of these results for the design and use of vaccines that provide temporary immunity.     

Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.     

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.     

Optical microscopy of growing insulin amyloid spherulites on surfaces in vitro

Amyloid fibrils are often found arranged into large ordered spheroid structures, known as spherulites, occurring in vivo and in vitro. The spherulites are predominantly composed of radially ordered amyloid fibrils, which self-assemble from protein in solution. We have observed and measured amyloid spherulites forming from heat-treated solutions of bovine insulin at low pH. The spherulites form in large numbers as semispherical dome-shaped objects on the cell surfaces, showing that surface defects or impurities, or the substrates themselves, can provide good nucleation sites for their formation. Using optical microscopy, we have measured the growth of individual spherulites as a function of time and in various conditions. There is a lag time before nucleation of the spherulites. Once they have nucleated, they grow, each with a radius increasing linearly, or faster than linearly, with time. Remarkably, this growth period has a sudden end, at which all spherulites in the system suddenly stop growing. A model of spherulite formation based on the polymerization of oriented fibrils around a nucleus, from a precursor in solution, quantitatively accounts for the observed growth kinetics. Seeding of native insulin solutions with preformed spherulites led to the preformed spherulites growing without a lag time. This seeding behavior is evidence that the fibrils in the spherulites assemble from small protein species rather than fibrils. The density of the spherulites was also measured and found to be constant with respect to radius, indicating that the space fills as the spherulite grows.     

Interleukin-17 acts independently of TNF-alpha under arthritic conditions

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.