A(3) adenosine receptor deficiency does not influence atherogenesis

Atherosclerosis is a multifactorial disease, the progression of which is modulated by several factors, including inflammation and hypercholesterolemia. The A(3) adenosine receptor (A(3)AR) has been reported to affect mast cell degranulation leading to inflammation, as well as to influence cardiovascular homeostasis. Here, we show that its deletion can also impact vascular smooth muscle cell (VSMC) proliferation in vitro. Based on these observations, we hypothesized that A(3)AR deficiency would affect atheromatous lesion development in vivo. Our results indicate that the expression of the matrix enzyme lysyl oxidase (LO) is increased while the proliferation potential of VSMC is decreased in A(3)AR-null aortas. This is in accordance with the previously reported inverse correlation between LO level and proliferation. Nevertheless, we found that A(3)-deficiency does not protect vessels against atherogenesis. This was demonstrated in mouse models of high fat diet-induced atherosclerosis and guidewire-induced femoral artery injury. We conclude that the contributions of the A(3)AR to inflammation and to modulating LO levels are not significant enough to control vascular response to injury.     

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.     

Habitat complexity: approaches and future directions

Habitat complexity is one of the most important factors structuring biotic assemblages, yet we still lack basic understanding of the underlying mechanisms. Although it is one of the primary targets in conservation management, no methods are available for comparing complexity across ecosystems, and system-specific qualitative assessment predominates. Despite its overwhelming importance for faunal diversity and abundance, there has been surprisingly little interest in examining its effects on other community and ecosystem attributes. We discuss possibilities of such effects, outlining potentially fruitful areas for future research, and argue that complexity may be implicated in community persistence and ecosystem stability by acting as a decoupling mechanism in predator–prey interactions. We provide a brief overview of methods used to quantify complexity in different ecosystems, highlighting contributions of the current issue of Hydrobiologia, and discuss potential application of these approaches for cross-ecosystem comparisons. Better understanding of the role of habitat complexity resulting from such comparisons is critically important for preservation of biodiversity and ecosystem function in an era of unprecedented habitat loss.     

New Roles for Cyclin E in Megakaryocytic Polyploidization

Megakaryocytes are platelet precursor cells that undergo endomitosis. During this process, repeated rounds of DNA synthesis are characterized by lack of late anaphase and cytokinesis. Physiologically, the majority of the polyploid megakaryocytes in the bone marrow are cell cycle arrested. As previously reported, cyclin E is essential for megakaryocyte polyploidy; however, it has remained unclear whether up-regulated cyclin E is an inducer of polyploidy in vivo. We found that cyclin E is up-regulated upon stimulation of primary megakaryocytes by thrombopoietin. Transgenic mice in which elevated cyclin E expression is targeted to megakaryocytes display an increased ploidy profile. Examination of S phase markers, specifically proliferating cell nuclear antigen, cyclin A, and 5-bromo-2-deoxyuridine reveals that cyclin E promotes progression to S phase and cell cycling. Interestingly, analysis of Cdc6 and Mcm2 indicates that cyclin E mediates its effect by promoting the expression of components of the pre-replication complex. Furthermore, we show that up-regulated cyclin E results in the up-regulation of cyclin B1 levels, suggesting an additional mechanism of cyclin E-mediated ploidy increase. These findings define a key role for cyclin E in promoting megakaryocyte entry into S phase and hence, increase in the number of cell cycling cells and in augmenting polyploidization.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Growth form evolution and hybridization in Senecio (Asteraceae) from the high equatorial Andes

Changes in growth forms frequently accompany plant adaptive radiations, including páramo–a high‐elevation treeless habitat type of the northern Andes. We tested whether diverse group of Senecio inhabiting montane forests and páramo represented such growth form changes. We also investigated the role of Andean geography and environment in structuring genetic variation of this group. We sampled 108 populations and 28 species of Senecio (focusing on species from former genera Lasiocephalus and Culcitium) and analyzed their genetic relationships and patterns of intraspecific variation using DNA fingerprinting (AFLPs) and nuclear DNA sequences (ITS). We partitioned genetic variation into environmental and geographical components. ITS‐based phylogeny supported monophyly of a Lasiocephalus‐Culcitium clade. A grade of herbaceous alpine Senecio species subtended the Lasiocephalus‐Culcitium clade suggesting a change from the herbaceous to the woody growth form. Both ITS sequences and the AFLPs separated a group composed of the majority of páramo subshrubs from other group(s) comprising both forest and páramo species of various growth forms. These morphologically variable group(s) further split into clades encompassing both the páramo subshrubs and forest lianas, indicating independent switches among the growth forms and habitats. The finest AFLP genetic structure corresponded to morphologically delimited species except in two independent cases in which patterns of genetic variation instead reflected geography. Several morphologically variable species were genetically admixed, which suggests possible hybrid origins. Latitude and longitude accounted for 5%–8% of genetic variation in each of three AFLP groups, while the proportion of variation attributed to environment varied between 8% and 31% among them. A change from the herbaceous to the woody growth form is suggested for species of high‐elevation Andean Senecio. Independent switches between habitats and growth forms likely occurred within the group. Hybridization likely played an important role in species diversification.     

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2^V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.     

Fish oil supplementation in F1 generation associated with naproxen, clenbuterol, and insulin administration reduce tumor growth and cachexia in Walker 256 tumor-bearing rats

Weanling female Wistar rats were supplemented with fish oil (1 g/kg body weight) for one generation. The male offspring received the same supplementation until to adult age. Rats supplemented with coconut fat were used as reference. Some rats were inoculated subcutaneously with a suspension (2 x 10(7) cells/mL) of Walker 256 tumor. At day 3, when the tumor was palpable, rats were treated with naproxen (N) (0.1 mg/mL), clenbuterol (Cb) (0.15 mg/kg body weight), and insulin (I) (10 U/kg body weight). At day 14 after tumor inoculation, the animals were killed. Tumor was removed and weighed. Blood, liver, and skeletal muscles were also collected for measurements of metabolites and insulin. In both tumor-bearing untreated rats and tumor-bearing rats supplemented with coconut fat, tumor growth, triacylglycerol, and blood lactate levels were higher, and glycogen content of the liver, blood glucose, cholesterol and HDL-cholesterol levels were lower as compared with the non-tumor-bearing and fish oil supplemented groups. Fish oil supplementation of tumor-bearing rats led to a partial recovery of the glycogen content in the liver and a full reversion of blood glucose, lactate, cholesterol, and HDL-cholesterol levels. The treatment with N plus Cb plus I attenuated cancer cachexia and decreased tumor growth in both coconut fat and fish oil supplemented rats. In conclusion, chronic fish oil supplementation decreased tumor growth and partially recovered cachexia. This beneficial effect of fish oil supplementation was potentiated by treatment with naproxen plus clenbuterol plus insulin.     

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.