Genetic predisposition and environmental factors contribute to an individual's susceptibility to develop hepatosteatosis. In a systematic, comparative survey we focused on genotype-dependent and -independent adaptations early in the pathogenesis of hepatosteatosis by characterizing C3HeB/FeJ, C57BL/6NTac, C57BL/6J, and 129P2/OlaHsd mice after 7, 14, or 21 days high-fat-diet exposure. Strain-specific metabolic responses during diet challenge and liver transcript signatures in mild hepatosteatosis outline the suitability of particular strains for investigating the relationship between hepatocellular lipid content and inflammation, glucose homeostasis, insulin action, or organelle physiology. Genetic background-independent transcriptional adaptations in liver paralleling hepatosteatosis suggest an early increase in the organ's vulnerability to oxidative stress damage what could advance hepatosteatosis to steatohepatitis. "Universal" adaptations in transcript signatures and transcription factor regulation in liver link insulin resistance, type 2 diabetes mellitus, cancer, and thyroid hormone metabolism with hepatosteatosis, hence, facilitating the search for novel molecular mechanisms potentially implicated in the pathogenesis of human non-alcoholic-fatty-liver-disease.

Key points Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin‐ and dysferlin‐deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin‐deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. Abstract Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin‐ and dysferlin‐deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β‐dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin‐deficient mice. Single‐unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp‐and‐hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly "N-labeled and 15N,'3C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 'H, '?N, and "C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional '5N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments. 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-I5N-edited and 'T-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a threehelix bundle and a small four-stranded antiparallel P-sheet that forms a hydrophobic core. The two Cterminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between p3 and p4. Thir leads to a tight turn between these p-strands.Keywords: NMR ; protein solution structure; DNA binding; heat-stress trancnption factor A central element of the rapid reprogramming of gene expression during the heat-stress response in all organisms is the activation of heat-stress genes. Newly formed heat-stress proteins are essential for the maintenance of the structural and functional integrity of cells during the stress period (Morimoto, 1993 ;Nover, 1991). With very few exceptions, heat-stress genes are characterized by a conserved palindromic recognition site in their promoter regions (5'-AGAANNTTCT-3'). The search for the corresponding transcription-activating proteins (heat-stress transcription factors, HSF) led to the identification of a new family of regulatory proteins with a N-terminal DNA-binding domain of about 100 amino acid residues which is conserved in yeast, insects, mammals and plants. (Scharf et al., 1990(Scharf et al., , 1994.Recently the three-dimensional structure of recombinant protein fragments of the yeast and Drosophila HSF has been investigated by means of X-ray crystallography (Harrison et al., 1994) and NMR techniques (Damberger et al., 1994; Vuister et al., 1994a,b). The DNA-binding domain contains a central helix-turn-helix motif which is thought to be responsible for specific DNA recognition (Vuister et al., 1994a Ahhrevinrions. DQF-COSY, double-quantum filtered correlation spectroscopy ; HMQC, heteronuclear-multiple-quantum-correlation spectroscopy ; HSE, heat-stress element; HSF, heat-stress transcription factor; HSQC, heteronuclear-single-quantum-correlation spectroscopy; TPPI, time-proportional phase incrementation.Enzyme. Thrombin (EC 3.4.21.5).remarkable similarities of all HSF, the three tomato proteins sequenced (Scharf et al...

ObjectiveExcess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance.MethodsWe induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet.ResultsExcess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver.ConclusionsWe assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties – in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.