Bim inhibits autophagy by recruiting beclin 1 to microtubules

Bim is a proapoptotic BH3-only Bcl-2 family member.?In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in?vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.     

In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2) were also reduced. Importantly, TGF-β(2) failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.     

Development of a biomimetic nanoporous membrane for the selective transport of charged proteins

We report the use of a micrometer-thick platinum-coated nanoporous membrane for the separation of differently charged proteins. A high field strength of about 25 kV m(-1) was applied, using very low transmembrane potentials of +/-1.5 V between the platinum-coated membranes. The system mimics the cell membrane function of facilitated transport for specific solutes. The selectivity for Lys:BSA:Mb in a mixed protein solution could be tuned readily between the flux ratios of 2:2:1 and 96:1:12 respectively, by simple variation of the transmembrane potentials from +1.5 V to -1.5 V. The experimental fluxes agreed closely with calculated fluxes derived from a simple electrophoresis-potential shielding model at favourable transmembrane potentials.     

BRCA1 and BRCA2 germline mutations in Malaysian women with early-onset breast cancer without a family history

Background

In Asia, breast cancer is characterised by an early age of onset: In Malaysia, approximately 50% of cases occur in women under the age of 50 years. A proportion of these cases may be attributable, at least in part, to genetic components, but to date, the contribution of genetic components to breast cancer in many of Malaysia''s ethnic groups has not been well-characterised.

Methodology

Given that hereditary breast carcinoma is primarily due to germline mutations in one of two breast cancer susceptibility genes, BRCA1 and BRCA2, we have characterised the spectrum of BRCA mutations in a cohort of 37 individuals with early-onset disease (≤40 years) and no reported family history. Mutational analysis of BRCA1 and BRCA2 was conducted by full sequencing of all exons and intron-exon junctions.

Conclusions

Here, we report a total of 14 BRCA1 and 17 BRCA2 sequence alterations, of which eight are novel (3 BRCA1 and 5 BRCA2). One deleterious BRCA1 mutation and 2 deleterious BRCA2 mutations, all of which are novel mutations, were identified in 3 of 37 individuals. This represents a prevalence of 2.7% and 5.4% respectively, which is consistent with other studies in other Asian ethnic groups (4–9%).     

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.     

Recruitment of thioredoxin-like domains into prostaglandin synthases

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently. We have identified two new enzymes, thioredoxin-type PGF synthase and membrane-associated PGE synthase-2, with thioredoxin-like domains. In addition, the N-terminal domain of hematopoietic PGD synthase is classified into the thioredoxin-like superfamily, based on structural similarity. The active sites of the former two enzymes have a CXXC motif, which is also critical for the thioredoxin activity. In contrast, hematopoietic PGD synthase lacks the motif, and the activity is carried out by glutathione. A phylogenetic tree of the thioredoxin-like domains suggests that they have been independently recruited into these PG synthases. We will discuss the functional meaning of the thioredoxin-like domains in the PG synthases from the viewpoint of the redox activity.     

Phototropism in Dunaliella and its application in a harvesting device.

Summary The influence of wavelength, light intensity and algal concentration on the phototropism of Dunaliella sp. is described. A practical device for harvesting the alga, based in this effect is shown.     

The fine structure of the lateral ocellus of the dobsonfly larva

The lateral ocelli of the dobsonfly (Protohermes grandis, Neuroptera) larva have been examined with light and electron microscopy. The larva has six ocelli on both sides of the head, each containing a single corneal lens. A conical crystalline body, of some 10–20 cells is situated immediately posterior to the lens. From 100 to 300 elongated retinular cells are arranged perpendicular to the crystalline body except at the innermost surface of the lens, where they are absent. The distal process of each retinular cell is enclosed by a tube-like rhabdom formed by the close association of microvilli from the same and adjacent distal processes. The distal process contains many mitochondria, multivesicular bodies, microtubles and pigment granules. In the dark-adapted ocellus the pigment granules are concentrated near the nucleus which lies under the rhabdomic layer. The granules diffuse toward the rhabdomic microvilli during light adaptation. Each retinular cell has a single axon, which extends from the ocellus as an ocellar nerve fiber into the optic lobe, where it frequently synapses upon second order neurons. In addition to these afferent synapses, there are two other synaptic combinations: (1) a feedback synapse from a second order neuron to a retinular axon, and (2) a synapse between second order neurons. These results suggest that photic signals reach the more proximal part of the brain via second order neurons after some degree of integration in the optic lobe.     

Soy‐Derived Phytochemical Genistein Modifies Chromatome Topology to Restrict Cancer Cell Proliferation

Epidemiological data indicate that human cancer risk is significantly reduced by the consumption of soy‐based foods containing the “phytoestrogen” genistein, which can signal via host cell estrogen receptors. While additional chemoprotective effects of genistein induced by epigenetic factors have also been reported, the key molecules and mechanisms involved are poorly defined. We therefore investigated genistein effects on chromatin‐bound proteins in the estrogen receptor‐deficient cell line MDA‐MB‐231 which is insensitive to phytoestrogen signaling. After exposure to low‐dose genistein for >1 month, MDA‐MB‐231 cells exhibited stable epigenetic alterations that are analyzed via partial MNase digestion and TMT‐based quantitative proteomics. 3177 chromatin‐bound proteins are identified with high confidence, including 882 molecules that displayed altered binding topology after cell conditioning with genistein. Prolonged phytochemical exposure conferred heritable changes in the binding topology of key epigenetic regulators including ATRX, SUV39H1/H2, and HP1BP3 that are preserved in untreated progeny, resulting in sustained downregulation of proliferation genes and reduced cell growth. These data indicate that soy derivative genistein exerts complex estrogen receptor‐independent effects on the epigenome likely to influence tumorigenesis by restricting cell growth.