DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors.     

An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 μs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.     

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.     

Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides

The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.     

Neospora caninum is associated with abortion in Algerian cattle

Neospora caninum is a major cause of abortion in cattle worldwide. However, little information is available for Algeria. Accordingly, 799 cattle from 87 farms in the north and northeast of Algeria were enrolled in a seroepidemiological survey. An indirect fluorescence antibody test (IFAT) revealed a seroprevalence of 19.6%. The animals were divided into 3 groups according to their breed: imported European cattle, local breeds, and crossed animals (European × local). Seroprevalences were 16.0%, 34.3%, and 18.6% in groups 1, 2, and 3, respectively. A case control study was performed to investigate the link between global seropositivity to N. caninum and abortion risk in those cattle farms. There was a significant (P < 0.01) association between the seroprevalence against N. caninum and the occurrence of abortion in those farms (odds ratio [OR] = 12.03). This was also observed at the individual level (OR = 2.79). The analysis of results according to the breed revealed a significant association between seroprevalence and abortion in groups 1 and 3, but not for group 2, despite the fact that the highest seroprevalence was observed in group 2. Cerebral tissues from 5 aborted fetuses were available for histology and polymerase chain reaction (PCR). One sample was found positive both by histology and by PCR, 2 samples were positive by PCR only, and 2 samples were negative in both tests.     

In CK2 inactivated cells the cyclin dependent kinase inhibitor Sic1 is involved in cell-cycle arrest before the onset of S phase

Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (alpha and alpha') are encoded by the CKA1, CKA2 genes. cka1Deltacka2(ts) mutants arrest cell cycle in both G1 and G2/M at 37 degrees C. Hence, it has been proposed that CK2 plays an important role in cell-cycle progression and several cell-cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5-Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5-Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells.     

Histological and immunohistological aspects of the ovarian cycle of the algerian wild sand rat, Psammomys obesus Cretzschmar, 1828

The sand rat, Psammomys obesus, is largely used as a model for studying several metabolic disorders. In order to perform breeding laboratory conditions, the reproductive function of this species was investigated. Using histological and immunohistochemical techniques, several aspects of the ovaries were studied throughout the sexual cycle. During the ovarian cycle, the different stages of folliculogenesis, from primordial to Graafian follicle, have been shown; the differentiation of both granulosa and theca cells, the formation of the antrum, cumulus oophorus and corona radiate were described. Broken follicles and corpora lutea have been observed, confirming a spontaneous ovulation in isolated females. Steroid activities were analysed using immunohistochemical techniques. Estrogen, androgen and progesterone hormones were visualized in the different compartments of the ovary.     

Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.     

Characterization of an extracellular polysaccharide produced by a Saharan bacterium Paenibacillus tarimensis REG 0201M

The purpose of this paper is to highlight the novel molecular characteristics and rheological properties of the polysaccharide produced by a bacterium isolated from extremophilic environment (Algerian Sahara). Phenotypic and molecular characteristics of the REG 0201M bacterial strain retained for this research were studied. Extracellular polysaccharide produced by REG 0201M was purified, characterized and its production was investigated. Analysis of 16S rDNA gene sequence showed that strain REG 0201M belonged to the species Paenibacillus tarimensis. In sucrose agar medium, 1.31 g dry weight of the polysaccharide per liter was produced. Evaluation of water absorption capacity showed that this polysaccharide was able to absorb 1000 times more water than its own weight. The average molecular weight determined by high-performance size exclusion chromatography multiangle laser light scattering was 1.718 × 106 g mol−1. Analysis of the monosaccharide composition by high-performance anion exchange chromatography showed the presence of fructose (77.67%) as the main neutral sugar, followed by galactose (20.37%), arabinose (1.79%), and rhamnose (0.16%). Its global charge determined by the Zeta potential measurement was about −35.27 ± 0.66 mV. The main functional groups were elucidated by Fourier-Transform Infrared Spectroscopy while the surface morphology was resolved by scanning electron microscopy analysis. Moreover, the rheological data revealed shear thinning properties with the same general behavior of the studied polysaccharide compared to xanthan. These results show the great potential of this polysaccharide, which could help to promote its use in various industries by replacing synthetic polymers.