Identification and pathway analysis of microRNAs with no previous involvement in breast cancer

microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described.     

Insulin Sensitivity Determines the Effectiveness of Dietary Macronutrient Composition on Weight Loss in Obese Women

Objective: To determine whether macronutrient composition of a hypocaloric diet can enhance its effectiveness and whether insulin sensitivity (Si) affects the response to hypocaloric diets. Research Methods and Procedures: Obese nondiabetic insulin‐sensitive (fasting insulin < 10 μU/mL; n = 12) and obese nondiabetic insulin‐resistant (fasting insulin > 15 μU/mL; n = 9) women (23 to 53 years old) were randomized to either a high carbohydrate (CHO) (HC)/low fat (LF) (60% CHO, 20% fat) or low CHO (LC)/high fat (HF) (40% CHO, 40% fat) hypocaloric diet. Primary outcome measures after a 16‐week dietary intervention were: changes in body weight (BW), Si, resting metabolic rate, and fasting lipids. Results: Insulin‐sensitive women on the HC/LF diet lost 13.5 ± 1.2% (p < 0.001) of their initial BW, whereas those on the LC/HF diet lost 6.8 ± 1.2% (p < 0.001; p < 0.002 between the groups). In contrast, among the insulin‐resistant women, those on the LC/HF diet lost 13.4 ± 1.3% (p < 0.001) of their initial BW as compared with 8.5 ± 1.4% (p < 0.001) lost by those on the HC/LF diet (p < 0.04 between two groups). These differences could not be explained by changes in resting metabolic rate, activity, or intake. Overall, changes in Si were associated with the degree of weight loss (r = ?0.57, p < 0.05). Discussion: The state of Si determines the effectiveness of macronutrient composition of hypocaloric diets in obese women. For maximal benefit, the macronutrient composition of a hypocaloric diet may need to be adjusted to correspond to the state of Si.     

Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate

Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni . During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase ( aspA ) mutant (unable to utilize any amino acid except serine) and a Cj0762 c ( aspB ) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA -dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.     

Phylogeny of Culturable Estuarine Bacteria Catabolizing Riverine Organic Matter in the Northern Baltic Sea

The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions. Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields. At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments. Five species in the Cytophaga-Flexibacter-Bacteroides group and one in the γ-proteobacteria phylogenetic group (Marinomonas sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures. Four of the isolates in Cytophaga-Flexibacter-Bacteroides group were putatively affiliated with the genus Flavobacterium. All dominating isolates were determined to be new species based on comparison to the current databases. The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary. It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species. Taken together, our results suggest that a limited group of bacteria, mainly in the Flavobacterium genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.     

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.     

Upstream and intronic regulatory sequences interact in the activation of the glutamine synthetase promoter.

Glutamine synthetase (GS) is expressed at high levels in subsets of cells in some tissues and at low levels in all cells of other tissues, suggesting that the GS gene is surrounded by multiple regulatory elements. We searched for such elements in the 2.5-kb upstream region and in the 2.6-kb first intron of the GS gene, using FTO-2B hepatoma and C2/7 muscle cells as representatives of both cell types and transient transfection assays as our tools. In addition to the entire upstream region and entire intron, an upstream enhancer module at -2.5 kb, and 5', middle and 3' modules of the first intron were tested. The main effects of the respective modules and their combinatorial interactions were quantified using the analysis of variance (anova) technique. The upstream enhancer was strongly stimulatory, the middle intron module strongly inhibitory, and the 3'-intron module weakly stimulatory in both hepatoma and muscle cells. The 5'-intron module was strongly stimulatory in muscle cells only. The major new finding was that in both cell types, the upstream enhancer and 5'-intron module needed to be present simultaneously to fully realize their transactivational potencies. This interaction was responsible for a pronounced inhibitory effect of the 5'-intron module in the absence of the upstream enhancer in hepatoma cells, and for a strong synergistic effect of these two modules, when present simultaneously in muscle cells. The main difference between hepatoma and muscle cells therefore appeared to reside in tissue-specific differences in activity of the respective regulatory elements due to interactions rather than in the existence of tissue-specific regulatory elements.