ESCRT proteins and cell signalling

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

Elevated CpG island methylation of GCK gene predicts the risk of type 2 diabetes in Chinese males

Background

The GCK gene encodes hexokinase 4, which catalyzes the first step in most glucose metabolism pathways. The purpose of our study is to assess the contribution of GCK methylation to type 2 diabetes (T2D).

Methods and results

GCK methylation was evaluated in 48 T2D cases and 48 age- and gender-matched controls using the bisulphite pyrosequencing technology. Among the four CpG sites in the methylation assay, CpG4 and the other three CpGs (CpG1-3) were not in high correlation (r < 0.5). Significantly elevated methylation levels of GCK CpG4 methylation were observed in T2D patients than in the healthy controls (P = 0.004). A breakdown analysis by gender indicated that the association between CpG4 methylation and T2D was specific to males (P = 0.002). It is intriguing that another significant male-specific association was also found between GCK CpG4 methylation and total cholesterol (TC) concentration (r = 0.304, P = 0.036).

Conclusion

Our results showed that elevated GCK CpG4 methylation might suggest a risk of T2D in Chinese males. Gender disparity in GCK CpG4 methylation might provide a clue to elaborate the pathogenesis of T2D.     

Activation of p38 Mitogen-Activated Protein Kinase in Gaucher’s Disease

Gaucher’s disease is caused by defects in acid β-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher’s disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher’s disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher’s disease mice. Most interestingly, neuronopathic Gaucher’s disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher’s disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher’s disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher’s disease mice. In mouse Gaucher’s disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher’s disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher’s disease.     

HN1L promotes migration and invasion of breast cancer by up-regulating the expression of HMGB1

Recent reports showed that haematological and neurological expressed 1-like (HN1L) gene participated in tumorigenesis and tumour invasion. However, the expression and role of HN1L in breast cancer remain to be investigated. Here, bioinformatics, western blot and immunohistochemistry were used to detect the expression of HN1L in breast cancer. Wound healing, transwell assay, immunofluorescence assay and mass spectrum were used to explore the role and mechanism of HN1L on the migration and invasion of breast cancer, which was confirmed in vivo using a nude mice model. Results showed that HN1L was significantly over-expressed in breast cancer tissues, which was positively correlated with M metastasis of breast cancer patients. Silencing HN1L significantly inhibited the invasion and metastasis of breast cancer cells in vitro and lung metastasis in nude mice metastasis model of breast cancer. Mechanistically, HN1L interacted with HSPA9 and affected the expression of HMGB1, playing a key role in promoting the invasion and metastasis of breast cancer cell. These results suggested that HN1L was an appealing drug target for breast cancer.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.     

The role of salicylic acid and carrot embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda)

Enhanced somatic embryogenesis and plant regeneration have been obtained using young leaf bases of naked oat (Avena nuda) as explants by including salicylic acid (SA) and carrot embryogenic callus extracts (CECE) in media. A 5- and 4-fold improvement was achieved in somatic embryogenesis and plant regeneration on the corresponding media supplemented with 0.5 mM SA and CECE as compared to control, respectively. Some physiological and biochemical changes were assayed in both embryogenic callus (EC) and non-embryogenic callus (NEC). The results indicated that superoxide dismutase activity was stimulated and catalases and ascorbate peroxidase activities were inhibited, while the O₂ ^- (superoxide anion) content was reduced and the hydrogen peroxide level was promoted in EC compared with NEC. Reduced malondialdehyde content and relative electrolyte leakage were also detected in EC.