Enhanced ABCG1 expression increases atherosclerosis in LDLr-KO mice on a western diet

ABCG1 promotes cholesterol efflux from cells, but ABCG1(-/-) bone marrow transplant into ApoE(-/-) and LDLr(-/-) mice reduces atherosclerosis. To further investigate the role of ABCG1 in atherosclerosis, ABCG1 transgenic mice were crossed with LDLr-KO mice and placed on a high-fat western diet. Increased expression of ABCG1 mRNA was detected in liver (1.8-fold) and macrophages (2.7-fold), and cholesterol efflux from macrophages to HDL was also increased (1.4-fold) in ABCG1xLDLr-KO vs. LDLr-KO mice. No major differences were observed in total plasma lipids. However, cholesterol in the IDL-LDL size range was increased by approximately 50% in ABCG1xLDLr-KO mice compared to LDLr-KO mice. Atherosclerosis increased by 39% (10.1+/-0.8 vs 6.1+/-0.9% lesion area, p=0.02), as measured by en face analysis, and by 53% (221+/-98 vs 104+/-58x10(3)microm(2), p =0.01), as measured by cross-sectional analysis in ABCG1xLDLr-KO mice. Plasma levels for MCP-1 (1.5-fold) and TNF-alpha (1.2-fold) were also increased in ABCG1xLDLr-KO mice. In summary, these findings suggest that enhanced expression of ABCG1 increases atherosclerosis in LDLr-KO mice, despite its role in promoting cholesterol efflux from cells.     

The use of pressure-jump relaxation kinetics to study protein folding landscapes

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.     

Bacterial tyrosinases

Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are important factors in wound healing and primary immune response. Their active site is characterized by a pair of antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a "type 3 copper centre" is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase). The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is still not completely understood, because nothing is known about their tertiary structure. One main reason for this deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-modified monomeric proteins with a low molecular mass of ca. 30kDa. They are secreted to the surrounding medium, where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is essential for the correct expression of the enzyme. This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get more insights in structure, enzymatic reactions and functions of "type 3 copper" proteins in general.     

Quantitative trait locus analysis for obesity reveals multiple networks of interacting loci

Obesity is a highly heritable and genetically complex trait with hundreds of potential loci identified. An intercross of 513 F₂ progeny between the SM/J × NZB/BINJ inbred mouse strains was generated to identify quantitative trait loci (QTL) that are involved in the weight of four fat pads: mesenteric, inguinal, gonadal, and retroperitoneal. Sex and lean body weight were treated as covariates in the analysis of these fat pads. This analysis uncoupled genetic effects related to overall body size from those influencing the adiposity of a mouse. We identified multiple significant QTL. QTL alleles associated with increased lean body weight and individual fat pad weights are contributed by the NZB background. Adiposity loci are distinct from these body size QTLs and high-adiposity alleles are contributed by the SM background. An extended network of epistatic QTL is also observed. A QTL on Chr 19 is the center of a network of eight interacting QTL, Chr 4 is the center of six, and Chr 17 the center of four interacting QTL. We conclude that interacting networks of multiple genes characterize the regulation of fat pad depots and body weight. Haplotype patterns and a literature-driven approach were used to generate hypotheses regarding the identity of the genes and pathways underlying the QTL.     

Genetic bases of renal agenesis in the ACI rat: mapping of Renag1 to chromosome 14

Unilateral renal agenesis (URA) is a common developmental defect in humans, occurring at a frequency of approximately 1 in 500–1000 births. Several genetic syndromes include bilateral or unilateral renal agenesis as an associated phenotype. However, URA frequently occurs in individuals not afflicted by these syndromes and is often asymptomatic. Although it is clear that genetic factors contribute to the etiology of URA, the genetic bases of URA are poorly defined at this time. ACI rats, both males and females, exhibit URA at an incidence of 5%–15%. In this article we characterize the incidence of URA in female and male F₁, F₂, and backcross (BC) progeny from reciprocal genetic crosses between the ACI strain and the unaffected Brown Norway (BN) strain. Through interval mapping analyses of 353 phenotypically defined female F₂ progeny, we mapped to rat Chromosome 14 (RNO14) a genetic locus, designated Renag1 (Renal agenesis 1), that serves as the major determinant of URA in these crosses. Further genotypic analyses of URA-affected female and male F₂ and BC progeny localized Renag1 to a 14.4-Mb interval on RNO14 bounded by markers D14Rat50 and D14Rat12. The data from these genetic studies suggest that the ACI allele of Renag1 acts in an incompletely dominant and incompletely penetrant manner to confer URA. James D. Shull and Cynthia M. Lachel authors contributed equally to this work.     

The proneural gene ascl1a is required for endocrine differentiation and cell survival in the zebrafish adenohypophysis

Mammalian basic helix-loop-helix proteins of the achaete-scute family are proneural factors that, in addition to the central nervous system, are required for the differentiation of peripheral neurons and sensory cells, derivatives of the neural crest and placodal ectoderm. Here, in identifying the molecular nature of the pia mutation, we investigate the role of the zebrafish achaete-scute homologue ascl1a during development of the adenohypophysis, an endocrine derivative of the placodal ectoderm. Similar to mutants deficient in Fgf3 signaling from the adjacent ventral diencepahalon, pia mutants display failure of endocrine differentiation of all adenohypophyseal cell types. Shortly after the failed first phase of cell differentiation, the adenohypophysis of pia mutants displays a transient phase of cell death, which affects most, but not all adenohypophyseal cells. Surviving cells form a smaller pituitary rudiment, lack expression of specific adenohypophyseal marker genes (pit1, neurod), while expressing others (lim3, pitx3), and display an ultrastructure reminiscent of precursor cells. During normal development, ascl1a is expressed in the adenohypophysis and the adjacent diencephalon, the source of Fgf3 signals. However, chimera analyses show that ascl1a is required cell-autonomously in adenohypophyseal cells themselves. In fgf3 mutants, adenohypophyseal expression of ascl1a is absent, while implantation of Fgf3-soaked beads into pia mutants enhances ascl1a, but fails to rescue pit1 expression. Together, this suggests that Ascl1a might act downstream of diencephalic Fgf3 signaling to mediate some of the effects of Fgf3 on the developing adenohypophysis.     

Novel photoreception system in sponges? Unique transmission properties of the stalk spicules from the hexactinellid Hyalonemasieboldi

Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 microm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and 1310 nm can pass through the fibers and (ii) light below wavelengths of 615 nm and above 1310 nm is completely cut-off. The transmission efficiency is around 60% (measured at 1080-1100 nm [length of the fiber: 5 cm]). The spicules acts as sharp high- and low-pass filters, suggesting that these silica-based fibers might be involved in a photoreception system. This assumption is supported by the finding that sponges are provided with a bioluminescent system. It is hypothesized that the spicules/siliceous fibers might be involved in a photoreception system in these animals.