Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.

Shortcomings of previously reported preclinical models of nonalcoholic steatohepatitis (NASH) include inadequate methods used to induce disease and assess liver pathology. We have developed a dietary model of NASH displaying features observed clinically and methods for objectively assessing disease progression. Mice fed a diet containing 40% fat (of which ∼18% was trans fat), 22% fructose, and 2% cholesterol developed three stages of nonalcoholic fatty liver disease (steatosis, steatohepatitis with fibrosis, and cirrhosis) as assessed by histological and biochemical methods.

Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of full-length peptide hormones than found using cell lysis procedures.

Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones.

Glucagon-like peptide-1 in the rat brain: distribution of expression and functional implication.

Glucagon-like-peptide 1 (GLP-1) is expressed not only in gut endocrine cells, but also in cells in the caudal brainstem and taste buds. To better understand the functions of central GLP-1, GLP-1 expression was immunohistochemically profiled in normal rat brain and its distribution correlated with FOS induction following systemic administration of a GLP-1 receptor agonist, exendin-4. In the present study, only a small number of GLP-1-immunoreactive cell bodies were observed in the nucleus of the solitary tract (NTS).

A primary colonic crypt model enriched in enteroendocrine cells facilitates a peptidomic survey of regulated hormone secretion.

To enable the first physiologically relevant peptidomic survey of gastrointestinal tissue, we have developed a primary mouse colonic crypt model enriched for enteroendocrine L-cells. The cells in this model were phenotypically profiled using PCR-based techniques and showed peptide hormone and secretory and processing marker expression at mRNA levels that were increased relative to the parent tissue. Co-localization of glucagon-like peptide-1 and peptide YY, a characteristic feature of L-cells, was demonstrated by double label immunocytochemistry.

Efficient high-throughput discovery of large peptidic hormones and biomarkers.

A novel approach is presented for the simultaneous identification and relative quantification of secreted peptides, particularly those that have been historically difficult to analyze in a concerted manner. Peptides exceeding 60 residues with various degrees of post-translational modification were identified on a liquid chromatographic time scale. The approach demonstrates high efficiency pattern-based recognition analysis of complex neuroendocrine peptide sets and enables rapid identification of biomarkers from biological material.

The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon.

Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases.

Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243.

Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that causes melioidosis, an invasive disease of humans and animals. To address the response of this bacterium to iron-limiting conditions, we first performed a global transcriptional analysis of RNA extracted from bacteria grown under iron-limiting and iron-rich conditions by microarrays. We focused our study on those open reading frames (ORFs) induced under iron limitation, which encoded predicted proteins that could be involved in the biosynthesis and uptake of the siderophore malleobactin.

The Burkholderia cepacia fur gene: co-localization with omlA and absence of regulation by iron.

The ferric uptake regulator (Fur) functions as a transcription repressor of many genes in bacteria in response to iron, but the presence of a functional equivalent of this protein has not been demonstrated in Burkholderia cepacia. A segment of the Burkholderia pseudomallei fur gene was amplified using degenerate primers and used to identify chromosomal restriction fragments containing the entire fur genes of B. cepacia and B.