RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.     

Protective effect of curcumin and chlorophyllin against DNA mutation induced by cyclophosphamide or benzo[a]pyrene

The current study was carried out to evaluate the potency of curcumin and chlorophyllin as natural antioxidants to reduce the oxidative stress markers induced by cyclophosphamide (CP) and benzo[a]pyrene [B(a)P] which were used as free radical inducers. For this purpose, 126 male albino rats were used. The animals were assigned into 4 main groups: negative control group; oxidant-treated group (subdivided into two subgroups: cyclophosphamide-treated group and benzo[a]pyrene-treated group); curcumin-treated group; and chlorophyllin-treated group. Liver samples were collected after two days post the oxidant inoculation and at the end of the experimental period (10 weeks). These samples were examined for determination of liver microsomal malondialdehyde (MDA), DNA fragmentation, restriction fragment length polymorphism (RFLP) and 8-hydroxy deoxyguanosine (8-OHdG) concentration. Both CP and B(a)P caused increments in DNA fragmentation percentages, liver microsomal MDA, concentration of 8-OHdG and induced point mutation. Treatment of rats with either curcumin or chlorophyllin revealed lower DNA fragmentation percentages, liver microsomal MDA concentration, concentration of 8-OHdG and prevented induction of mutations, i.e., reversed the oxidative stress induced by CP and B(a)P and proved that they were capable of protecting rats against the oxidative damage evoked by these oxidants.     

Novel synthesis of [1]-benzothiepino[5,4-b]pyridine-3-carbonitriles and their anti-inflammatory properties

Reaction of 4-arylmethylene-3,4-dihydro-[1]-benzothiepin-5(2H)-ones 1 with malononitrile in the appropriate alcohol in the presence of sodium afforded the 2-alkoxy-4-aryl-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-3-carbonitriles 2 and not the isomeric forms [1]-benzothiepino[4,5-c]pyridine-1-carbonitriles 3 in high regioselective manner. The assumed structure of 2 was inferred through independent synthetic reaction of 3,4-dihydro-[1]-benzothiepin-5(2H)-one (4) with ylidenemalononitriles 5 under the same applied reaction conditions and confirmed by single crystal X-ray diffraction studies. However, reaction of 4 with arylidenecyanothioacetamides 6 in refluxing ethanol in the presence of basic catalyst (piperidine or morpholine) does not afford the expected 4-aryl-3-cyano-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-2(1H)-thiones 7 and instead 4-aryl-3,5-dicyano-6-thioxo-2(1H)-pyridinethiolate monohydrates were isolated as piperidinium or morpholinium salts 8. On the other hand, reaction of 6 with cyanothioacetamide in the presence of a sufficient amount of basic catalyst yielded exclusively 2-amino-4-aryl-3,5-dicyano-2-pyridinethiolates as piperidinium or morpholinium salts 9. Meanwhile, 7 were prepared through the reaction of 1 with cyanothioacetamide in refluxing ethanol in the presence of a catalytic amount of piperidine. Anti-inflammatory activity screening of the prepared compounds using in vivo acute carrageenan-induced paw oedema in rats exhibited that all the tested compounds possess considerable activity. In addition, few synthesized derivatives reveal remarkable anti-inflammatory properties (2d, k, l) comparable with indomethacin which was used as a reference standard during the pharmacological activity screening studies.     

Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.     

Reduced survivin expression and tumor cell survival during chronic hypoxia and further cytotoxic enhancement by the cyclooxygenase-2 inhibitor celecoxib

Hypoxia is a characteristic feature of advanced solid tumors and may worsen prognosis. The development of tumor-targeted and hypoxia-inducible gene therapy vectors holds promise to selectively deliver and express suicidal or cytotoxic genes in hypoxic regions of tumors. In this regard, the promoter of the survivin gene, which encodes an anti-apoptotic protein that is strongly expressed in tumor tissue, has received attention because of its supposed inducibility by hypoxia. However, in our present study we demonstrate that treatment of various tumor cell lines with chronic hypoxia or with the hypoxia-mimetic CoCl₂ does not result in increased expression of survivin, but rather strongly suppresses this gene’s activity. In contrast, expression of glucose-regulated protein 78 (GRP78/Bip) is substantially elevated under chronic hypoxia in vitro and in hypoxic areas of tumor tissue in vivo. Although tumor cells in general exhibit increased chemoresistance under hypoxic conditions, we found that hypoxic glioblastoma cells are more sensitive to killing by the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib, and this effect is reflected by further decreased expression of survivin. Intriguingly, 2,5-dimethyl-celecoxib (DMC), a close structural analog of celecoxib that lacks the ability to inhibit COX-2, is able to potently mimic the anti-tumor effects of its parent compound, indicating that inhibition of COX-2 is not involved in these processes. Taken together, our results caution against the use of survivin-based promoters to target hypoxic areas of tumors, but favor constructs that include the strongly hypoxia-inducible GRP78 promoter. In addition, our data introduce celecoxib as a drug with increased cytotoxicity against hypoxic tumor cells.     

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">simulans</Emphasis> lipase expressed in <Emphasis Type="Italic">E. coli</Emphasis>

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.     

Novel Synthesis,Reactions, and Biological Study of New Morpholino-Thieno[2,3-c][2,7]Naphthyridines as Anti-Cancer and Anti-Microbial Agents

Russian Journal of Bioorganic Chemistry - The biological uses of 2,7-naphthyridines have sparked great attention in recent years. For this reason, we synthesized a series of novel...     

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants,Studies in Fibroblasts and Review of Literature

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II?+?III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient’s cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

     

The Black-necked Grebe (Podiceps nigricollis): an expanding species in the Middle Atlas wetlands,Morocco

The present study investigates the population trends of Black-necked Grebe Podiceps nigricollis (Podicipedidae: Podicidae) over 5 years (2009–2013) in three Middle Atlas wetlands (Aguelmam Afennourir, Dayet Aoua and Dayet Ifrah). Using generalized linear models with a Poisson distribution, we demonstrated that the number of Black-necked Grebe breeding pairs has varied significantly and positively over the 5 years and between the three study wetlands. The annual population growth rate of the three colonies was 0.48 (±0.01). This positive trend is consistent with the current Least Concern conservation status of the IUCN Red List. Further more detailed studies are, however, needed to improve our understanding on the mechanisms driving the population increase in this part of North Africa. This remains a prerequisite for proper population conservation and management.     

Statistical Cluster Analysis of the British Thoracic Society Severe Refractory Asthma Registry: Clinical Outcomes and Phenotype Stability

Background

Severe refractory asthma is a heterogeneous disease. We sought to determine statistical clusters from the British Thoracic Society Severe refractory Asthma Registry and to examine cluster-specific outcomes and stability.

Methods

Factor analysis and statistical cluster modelling was undertaken to determine the number of clusters and their membership (N = 349). Cluster-specific outcomes were assessed after a median follow-up of 3 years. A classifier was programmed to determine cluster stability and was validated in an independent cohort of new patients recruited to the registry (n = 245).

Findings

Five clusters were identified. Cluster 1 (34%) were atopic with early onset disease, cluster 2 (21%) were obese with late onset disease, cluster 3 (15%) had the least severe disease, cluster 4 (15%) were the eosinophilic with late onset disease and cluster 5 (15%) had significant fixed airflow obstruction. At follow-up, the proportion of subjects treated with oral corticosteroids increased in all groups with an increase in body mass index. Exacerbation frequency decreased significantly in clusters 1, 2 and 4 and was associated with a significant fall in the peripheral blood eosinophil count in clusters 2 and 4. Stability of cluster membership at follow-up was 52% for the whole group with stability being best in cluster 2 (71%) and worst in cluster 4 (25%). In an independent validation cohort, the classifier identified the same 5 clusters with similar patient distribution and characteristics.

Interpretation

Statistical cluster analysis can identify distinct phenotypes with specific outcomes. Cluster membership can be determined using a classifier, but when treatment is optimised, cluster stability is poor.