EGFR and beta1 integrins utilize different signaling pathways to activate Akt

Akt, also called PKB, is a serine/threonine kinase that plays a major role in cell survival. It can be activated by several cellular receptors, including integrins and growth factor receptors, in PI3K-dependent manners. In this study, we analyzed the two current models for Akt activation upon beta1 integrin-mediated adhesion: via focal adhesion kinase and via transactivation of the EGF receptor. Distinct differences in the pathways leading to phosphorylation and activation of Akt from stimulated beta1 integrins and EGF receptor were observed, including opposing sensitivity to the tyrosine kinase inhibitors PP2 and Gefitinib. Using knockout cells and integrin mutant cells, we show that beta1 integrins can induce phosphorylation of Akt at Ser473 and Thr308 and Akt kinase activity independently of the EGF receptor activity, focal adhesion kinase, and the Src family members. In contrast to stimulation with EGF, beta1 integrin-mediated adhesion did not induce Akt tyrosine phosphorylation. Moreover, tyrosine phosphorylation of Akt was found not to be required for its catalytic activity. The results identify a previously unrecognized mechanism by which beta1 integrins activate the PI3K/Akt pathway.     

Persistent growth effects of temperature and photoperiod in Atlantic cod Gadus morhua

Short-term environmental manipulations during the early juvenile stage have a large impact on harvesting size of Atlantic cod Gadus morhua nearly 3 years later. A group of juvenile Atlantic cod (initial mass 9·5 g) were reared for 3 months under simulated natural photoperiod or continuous light, and a range of temperatures (7, 10, 13 and 16° C, and a group called T-step, i.e. with temperature reduced successively from 16 to 13 and 10° C). After termination of the laboratory trial, the fish were moved to sea pens and reared at ambient conditions for 30 months before harvesting in June 2006. Observed growth gain from the 3 month laboratory trial was still persistent following the 30 months of sea-pen ongrowing. The T-step group displayed 15, 13, 1 and 10% superior mass gain respectively than the groups initially at 7, 10, 13 and 16° C at harvest in June 2006. Similarly, rearing under continuous light during the initial 3 month period during the early juvenile stage resulted in 1–9% larger size at harvesting compared to fish reared at simulated natural photoperiod. Gonado-somatic and hepato-somatic indices were similar in all groups. Contribution to the understanding of the mechanism behind size variation in adult fish can have wide range applications for Atlantic cod fisheries and aquaculture.     

Branchial expression patterns of claudin isoforms in Atlantic salmon during seawater acclimation and smoltification

In euryhaline teleosts, permeability changes in gill epithelia are essential during acclimation to changed salinity. This study examined expression patterns of branchial tight junction proteins called claudins, which are important determinants of ion selectivity and general permeability in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI, and classification was performed with the aid of maximum likelihood analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b, and 30) were present, and quantitative PCR analysis confirmed tissue-specific expression in gill when compared with kidney, intestine, heart, muscle, brain, and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced the expression of claudin 27a and claudin 30 but had no overall effect on claudin 28a and claudin 28b. In contrast, SW induced a fourfold increase in expression of claudin 10e. In accord, a peak in branchial claudin 10e was observed during smoltification in May, coinciding with optimal SW tolerance. Smoltification induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e vs. claudin 27a and 30. It is possible that claudin 10e is an important component of cation selective channels, whereas reduction in claudin 27a and 30 may change permeability conditions in favor of the ion secretory mode of the SW gill.     

Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle

Protein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases. Here we used a phosphosite specific antibody to investigate the phosphorylation of I-2. We fractioned extracts from HeLa cells arrested with nocodazole and assayed for PXTP kinases using recombinant I-2. One major and two minor peaks of kinase activity were identified and the major peak contained both active MAPK and cdk1::cyclinB1, confirmed by immunoblotting. Cells released from a double thymidine block synchronously progressed through mitosis and immunoblotting revealed transient phosphorylation of endogenous I-2 in cells only during mitosis, and corresponding phosphorylation of histone H3 (Ser10) and PP1 (Thr320). Activation of cdk1::cyclinB1 was coincident with I-2 phosphorylation, but neither MAPK nor GSK3 were phosphorylated at this time, so we concluded that in living cells only cdk1::cyclinB1 phosphorylated the PXTP site in I-2. Immunofluorescent staining of cells with the PXTP phosphosite antibody revealed highly specific staining of mitotic cells prior to anaphase, at which point the staining disappeared. Thus, phosphorylation of I-2 is catalyzed by cdk1::cyclinB1 and staining with a specific antibody should prove useful as a selective marker of cells in the early stages of mitosis.     

Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes

Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 °C, whereas at 25 °C and 37 °C the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and -carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73–78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.Key words: BRCA1, methylation, epigenetics, triple negative, breast cancer, retionblastoma tumor suppressor gene, pRb, p16     

The Impact of Divergence Time on the Nature of Population Structure: An Example from Iceland

The Icelandic population has been sampled in many disease association studies, providing a strong motivation to understand the structure of this population and its ramifications for disease gene mapping. Previous work using 40 microsatellites showed that the Icelandic population is relatively homogeneous, but exhibits subtle population structure that can bias disease association statistics. Here, we show that regional geographic ancestries of individuals from Iceland can be distinguished using 292,289 autosomal single-nucleotide polymorphisms (SNPs). We further show that subpopulation differences are due to genetic drift since the settlement of Iceland 1100 years ago, and not to varying contributions from different ancestral populations. A consequence of the recent origin of Icelandic population structure is that allele frequency differences follow a null distribution devoid of outliers, so that the risk of false positive associations due to stratification is minimal. Our results highlight an important distinction between population differences attributable to recent drift and those arising from more ancient divergence, which has implications both for association studies and for efforts to detect natural selection using population differentiation.     

Effects of 3-thia fatty acids on feed intake, growth, tissue fatty acid composition, beta-oxidation and Na+,K+-ATPase activity in Atlantic salmon

Atlantic salmon (Salmo salar) with an initial mass of 86 g were reared in 12 °C seawater for 8 weeks to a final average mass of 250 g. The fish were fed fish meal and fish oil-based diet supplemented with either 0%, 0.3% or 0.6% of tetradecylthioacetic acid (TTA), a 3-thia fatty acid. The specific growth rate (SGR) decreased with increasing dietary dose of TTA. The SGR of the group fed 0% of TTA (Control) was 1.8; that of the group fed 0.3% of TTA (TTA-L) was 1.7, and that of the group fed 0.6% of TTA (TTA-H) was 1.5. The mortality increased with increased dietary dose of TTA. The mitochondrial β-oxidation capacity in the liver of fish fed the TTA diets was 1.5 to 2 times higher than that of the Control fish. TTA supplementation caused substantial changes in the fatty acid compositions of the phospholipids (PL), triacylglycerols (TAG) and free fatty acids (FFA) of gills, heart and liver. The percentages of n−3 fatty acids, particularly 22:6 n−3, increased in fish fed diets containing TTA, while the percentage of the saturated FAs 14:0 and 16:0 in the PL fractions of the gills and heart decreased. The sum of monounsaturated FAs in the PL and TAG fractions from liver was significantly higher in fish fed diets containing TTA. TTA itself was primarily incorporated into PL. Two catabolic products of TTA (sulphoxides of TTA) were identified, and these products were particularly abundant in the kidney. TTA supplementation had no significant effect on the activity of the membrane-bound enzyme Na⁺,K⁺-ATPase.