Molecular characterization of WFS1 in an Iranian family with Wolfram syndrome reveals a novel frameshift mutation associated with early symptoms

Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder that represents a likely source of childhood diabetes especially among countries in the consanguinity belt. The main responsible gene is WFS1 for which over one hundred mutations have been reported from different ethnic groups. The aim of this study was to identify the molecular etiology of WS and to perform a possible genotype–phenotype correlation in Iranian kindred.     

A communication network within the cytoplasmic domain of toll-like receptors has remained conserved during evolution

Toll/IL-1R (TIR) domain, that is, the cytoplasmic domain, in toll-like receptors (TLRs) from different species showed high sequence conservation in stretches spread across the surface as well as the core of the domain. To probe the structure–function significance of these residues, especially those coming from the core of TIR domains, we analyzed molecular dynamics trajectories of sequence similarity based models of human TIR domains. This study brought forth that N-terminal of the TIR domain simultaneously interacts with the flanking residues of the BB loop and central β-sheets. At the same time, residues of the central β-strands form favorable contacts with the DD loop and C-terminal, thus forming a two-way circuit between the N- and C-termini. In this work, the array of intradomain interactions is termed as communication network. Importantly, the “hubs” of this communication network were found to be conserved in all human TLRs. Earlier mutagenesis–function correlation work brought forth that certain mutations in the “core” of the TIR domain of TLR4 (e.g. in IFI767–769AAA and L815A) led to almost complete abrogation of signaling and reasoning for this dramatic loss-of-function has remained unclear, since these sites are not surface exposed. Using MD studies, we show here that this communication network gets disrupted in mutants of human TLR4 which were earlier reported to be functionally compromised. Extension of MD studies to heterodimer of TLR1/2 suggested that this evolutionarily conserved communication network senses the interactions formed upon dimerization and relays it to surfaces which are not involved in direct interdomain contacts.     

Host status of some broad bean,Vicia faba L., selections to reniform nematode,Rotylenchulus reniformis,under greenhouse conditions in Egypt

Twenty broad bean selections were screened for their relative susceptibility to the reniform nematode, Rotylenchulus reniformis, under greenhouse conditions at 20?±?5?°C. The degree of susceptibility was rated based on number of females per root system of each selection. Hence, G 3, L 47, L 49, L 52, L 57, L 71, L 92, L 99 and L 375 selections were graded as resistant hosts against the nematode infection and sex selections (G 429, L 13, L 16, L 24, L 31 and L 46) were categorised as moderately resistant. However, only the selection L 9 out of 20 selections was ranked as susceptible and four selections, namely L 50, L 101, L 241 and L 348, were rated as highly susceptible against R. reniformis infection. It was observed that reproduction of the nematode was favoured on highly susceptible and susceptible selections but inhibited on resistant ones. Therefore, all tested selections showed great variability in their reaction to the nematode infection according to the host type. Also, various plant growth parameters were discussed. Finally, the differences among the tested selections should serve as an informative resource for plant breeders and cropping systems to limit the loss due to the nematode infection.     

Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities

High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC) levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.     

SHP-2-Erk signaling regulates concanavalin A-dependent production of TIMP-2

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.     

Synthesis of asparagine-linked bacillosamine

Various types of protein glycosylation have been identified from prokaryotes. Recent investigations have revealed the presence of N-linked glycoproteins in the pathogenic bacterium, Campylobacter jejuni. The structure of this glycan is unique, consisting of 5 GalNAc and 1 Glc, in addition to 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose (bacillosamine; Bac), which is N-glycosidically linked to the side chain of asparagine (Asn). We synthesized Bac from a 2-azido-2-deoxy-D-galactose derivative, which was further converted to the Asn-linked form.     

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.     

Enhanced FTase activity achieved via piperazine interaction with catalytic zinc

Benzocycloheptapyridine tricyclic compounds with piperazine or substituted piperidine moieties extending either from the 5- or 6-position of the tricyclic bridgehead exhibited enhanced FTase activity: this resulted from favorable binding of the ligand nitrogen with the catalytic zinc found in the FTase. A single isomer at C-11 with piperazine adduct extending from the 6-position, compound 24, exhibited excellent FTase activity with IC50 = 0.007 microM, soft agar IC50 = 72 nM, and Rat AUC(PO, 10 mpk) = 4.0 microM x h. X-ray of (-)-[8-chloro-6-(1-piperazinyl)-1H-benzo[5,6]]cyclohepta[1,2-b]pyridine-11-yl]-1-(methylsulfonyl)piperidine 24 bound to Ftase revealed favorable interaction between piperazine nitrogen and catalytic zinc atom.     

ABCA2 is a marker of neural progenitors and neuronal subsets in the adult rodent brain

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin(+) cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.     

Aldose reductase mediates the lipopolysaccharide-induced release of inflammatory mediators in RAW264.7 murine macrophages

Abnormal production of inflammatory cytokines and chemokines is a key feature of bacterial endotoxin, lipopolysaccharide (LPS)-induced inflammation, and cytotoxicity; however, the mechanisms regulating production of inflammatory markers remain unclear. Herein, we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR; AKR1B3) modulates NF-kappaB-dependent activation of inflammatory cytokines and chemokines in mouse serum, liver, heart, and spleen. Pharmacological inhibition or small interfering RNA ablation of AR prevented the biosynthesis of tumor necrosis factor-alpha, interleukin 1beta, interleukin-6, macrophage-chemoattractant protein-1, and cyclooxygenase-2 and prostaglandin E(2) in LPS-activated RAW264.7 murine macrophages. The AR inhibition or ablation significantly attenuated LPS-induced activation of protein kinase C (PKC) and phospholipase C (PLC), nuclear translocation of NF-kappaB, and phosphorylation and proteolytic degradation of IkappaBalpha in macrophages. Furthermore, treatment of macrophages with 4-hydroxy-trans-2-nonenal (HNE), and cell-permeable esters of glutathionyl-4-hydroxynonanal (GS-HNE) and glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF-kappaB and PLC/PKC. Pharmacological inhibition or antisense ablation of AR that catalyzes the reduction of GS-HNE to GS-DHN prevented PLC, PKC, IKKalpha/beta, and NF-kappaB activation caused by HNE and GS-HNE, but not by GS-DHN, suggesting that reduced GS-lipid aldehydes catalyzed by AR propagate LPS-induced production of inflammatory markers. Collectively, these data provide evidence that inhibition of AR may be a significant therapeutic approach in preventing bacterial endotoxin-induced sepsis and tissue damage.