AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC50 = 0.8 microM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC50 = 3.2 microM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.

Nitric oxide synthase (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOSnegative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over IntroductionThe intracellular formation of nitric oxide (NO) has been extensively studied in various mammalian tissues. NO (1,2), or a labile intermediate that is able to release NO, is generated from a terminal guanidino nitrogen of L-arginine (3-5) by a family of NO synthases (NOS; EC 1.14.23). L-Citrulline is the byproduct of this metabolic pathway. NO is the first messenger molecule of a signal transduction pathway (Figure 1). Although other targets for NO Supported by research grants DK 30787 and H I . 28474 several non-neural cell types and tissues. These included glia cells, m a d densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker. (J Histochem Cytochem 40:1439-1456, 1992) KEY WORDS: Cyclic GMP; Brain; Pancreas; Kidney; Stomach; Lung; Uterus; Bone; Epithelium; Endometrium. exist (6), its main mechanism of action involves binding to and activation of soluble guanylyl cyclase (GC-S; GTP pyrophosphatelyase (cyclizing), EC 4.6.1.2) (7), which then forms the second messenger molecule guanosine 3',5'-cyclic monophosphate (cGMP).NOS have been purified and characterized from brain (8-14), macrophages (15,16), and endothelial cells (17), and recently also from human cerebellum (18). Molecular cloning has provided clear evidence that brain (19) and macrophage (20) NOS are products of different genes. The relation between brain and endothelial NOS is unclear. Another polydonal antibody raised against rat brain NOS (21) was reported to bind to endothelial cell matrix, and the authors suggested that brain and endothelial NOS are highly homologous if not identical (22). Conversely, endothelial NOS was reported by our laboratory to differ from brain NOS with respect to molecular mass and subcellular location (17) and we proposed a classification of at least three types...

The diversity of the microbial community on cow teat skin was evaluated using a culture-dependent method based on the use of different dairy-specific media, followed by the identification of isolates by 16S rRNA gene sequencing. This was combined with a direct molecular approach by cloning and 16S rRNA gene sequencing. This study highlighted the large diversity of the bacterial community that may be found on teat skin, where 79.8% of clones corresponded to various unidentified species as well as 66 identified species, mainly belonging to those commonly found in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea, Aerococcus, and Staphylococcus). Several of them, such as nonstarter lactic acid bacteria (NSLAB), Staphylococcus, and Actinobacteria, may contribute to the development of the sensory characteristics of cheese during ripening. Therefore, teat skin could be an interesting source or vector of biodiversity for milk. Variations of microbial counts and diversity between the farms studied have been observed. Moreover, Staphylococcus auricularis, Staphylococcus devriesei, Staphylococcus arlettae, Streptococcus bovis, Streptococcus equinus, Clavibacter michiganensis, Coprococcus catus, or Arthrobacter gandavensis commensal bacteria of teat skin and teat canal, as well as human skin, are not common in milk, suggesting that there is a breakdown of microbial flow from animal to milk. It would then be interesting to thoroughly study this microbial flow from teat to milk.

Engagement of the T cell antigen receptor (TcR)1 with the antigen-major histocompatibility complex on antigen-presenting cells triggers a complex TcR signaling cascade that leads to T cell activation and cytokine secretion (1). During this process, T cells express the autocrine growth factor interleukin 2 (IL-2), which promotes T cell proliferation by interacting with the IL-2 receptor, which is also up-regulated on activated T cells. The transcriptional regulation of the IL-2 gene has been extensively analyzed at the IL-2 promoter, a 275-bp region located upstream of the transcriptional start site of the gene (2, 3). Several transcription factors have been identified to bind elements within this regulatory region, including AP-1, NF-B, and the nuclear factor of activated T cells (NFAT) (2).The transcription factor NFAT plays an essential role in IL-2 expression. Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-3, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor ␣, granulocyte-macrophage colony-stimulating factor, and ␥-IFN (4, 5). NFAT is a complex composed of a cytoplasmic subunit and an inducible nuclear component comprised of AP-1 (Fos/ Jun) family members. At least four structurally related NFAT cytoplasmic subunit members, NFATp/NFAT1, NFATc/ NFAT2, NFAT3, and NFATX/NFATc3/NFAT4, have been identified (5). NFAT proteins share a conserved domain located toward the C terminus (6) that binds DNA and also participates in cooperative protein-protein interactions with AP-1 transcription factors (7,8). Immediately N-terminal to the DNA-binding domain is a second conserved module of ϳ300 residues known as the NFAT homology (NFAT-h) region. The N terminus of NFAT, including the NFAT-h region, regulates nuclear/cytoplasm trafficking in response to changes in intracellular Ca 2ϩ concentrations. In resting T cells, the protein is retained in the cytoplasm and its NFAT-h domain is heavily phosphorylated. Engagement of the TcR or treatment of cells with the Ca 2ϩ ionophore activates the Ca 2ϩ /calmodulin-dependent Ser/Thr phosphatase, calcineurin. CaN dephosphorylates the NFAT-h domain, resulting in translocation of NFAT to the nucleus (9).

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Using a reverse transcription PCR (RT‐PCR) amplification strategy, with degenerate primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, GPX‐encoding cDNA fragments were generated from sunflower (Helianthus annuus L.) hypocotyl RNA. Along with the sequences of two distinct GPX‐encoding cDNAs (GPXha‐1 and GPXha‐2) and the alignment of their conceptually translated products with to date cloned plant GPXs, we report here that the two corresponding mRNAs are qualitatively and quantitatively differentially expressed in various organs of H. annuus. Using northern blotting we have shown that more sunflower GPX‐like transcripts accumulate in plants which have been subjected to either wounding or infection by a biotrophic pathogen (Plasmopara halstedii) suggesting that the corresponding genes are inducible under various stress conditions.

We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria.

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