Modulation of apolipoprotein A1 and B, adiponectin, ghrelin, and growth hormone concentrations by plant sterols and exercise in previously sedentary humans

Plant sterols combined with exercise beneficially alter lipid levels in hypercholesterolemic adults. The effect of this combination therapy on other indicators of coronary heart disease risk, however, has yet to be determined. The objective of this trial was to investigate the effect of plant sterols and exercise, alone and in combination, on levels of apolipoprotein (apo) A1 and B, adiponectin, ghrelin, and growth hormone in previously sedentary hypercholesterolemic adults. In an 8 week, parallel-arm trial, 84 subjects were randomized to 1 of 4 groups: combination, exercise, plant sterols, or control. Body mass decreased by 1.1% (p < 0.01) and 0.9% (p < 0.05) in the combination and exercise group, respectively. Low-density lipoprotein cholesterol levels decreased (p < 0.01) by 0.30 mmol/L in the combination group and by 0.49 mmol/L in the plant sterol group. High-density lipoprotein cholesterol levels increased by 7.5% and 9.5% (p < 0.01) in the combination and exercise groups, respectively. Plant sterols increased (p < 0.05) adiponectin levels by 16%. No change in apoA1, apoB, ghrelin, or growth hormone levels were noted in any intervention group. ApoA1 was correlated with high-density lipoprotein cholesterol (r = 0.33, p = 0.01), whereas apoB was weakly related to low-density lipoprotein cholesterol levels (r = 0.13, p = 0.002). Adiponectin was associated with body mass index (r = -0.10, p = 0.006) and high-density lipoprotein cholesterol (r = 0.17, p = 0.0003). These findings suggest that plant sterols can increase adiponectin levels, thereby possibly reducing the risk of future coronary heart disease.     

Ecological niche and geographic distribution of human monkeypox in Africa

Monkeypox virus, a zoonotic member of the genus Orthopoxviridae, can cause a severe, smallpox-like illness in humans. Monkeypox virus is thought to be endemic to forested areas of western and Central Africa. Considerably more is known about human monkeypox disease occurrence than about natural sylvatic cycles of this virus in non-human animal hosts. We use human monkeypox case data from Africa for 1970-2003 in an ecological niche modeling framework to construct predictive models of the ecological requirements and geographic distribution of monkeypox virus across West and Central Africa. Tests of internal predictive ability using different subsets of input data show the model to be highly robust and suggest that the distinct phylogenetic lineages of monkeypox in West Africa and Central Africa occupy similar ecological niches. High mean annual precipitation and low elevations were shown to be highly correlated with human monkeypox disease occurrence. The synthetic picture of the potential geographic distribution of human monkeypox in Africa resulting from this study should support ongoing epidemiologic and ecological studies, as well as help to guide public health intervention strategies to areas at highest risk for human monkeypox.     

A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO₂ and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H⁺ and CO₂ to favor the metabolically preferred carboxylation product, acetoacetate.     

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.     

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.     

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.     

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.     

Fast-twitch skeletal muscles of dystrophic mouse pups are resistant to injury from acute mechanical stress

Loss of the dystrophin-glycoproteincomplex from muscle sarcolemma in Duchenne's muscular dystrophy (DMD)renders the membrane susceptible to mechanical injury, leaky toCa²⁺, and disrupts signaling, but the precise mechanism(s)leading to the onset of DMD remain unclear. To assess the role ofmechanical injury in the onset of DMD, extensor digitorum longus (EDL)muscles from C57 (control), mdx, andmdx-utrophin-deficient [mdx:utrn(/); dystrophic] pups aged 9-12 days were subjected to an acutestretch-injury or no-stretch protocol in vitro. Before the stretches,isometric stress was attenuated for mdx:utrn(/) comparedwith control muscles at all stimulation frequencies (P < 0.05). During the stretches, EDL muscles for each genotypedemonstrated similar mean stiffness values. After the stretches,isometric stress during a tetanus was decreased significantly for bothmdx and mdx:utrn(/) muscles compared withcontrol muscles (P < 0.05). Membrane injury assessedby uptake of procion orange dye was greater for dystrophic comparedwith control EDL (P < 0.05), but, within eachgenotype, the percentage of total cells taking up dye was not different for the no-stretch vs. stretch condition. These data suggest that thesarcolemma of maturing dystrophic EDL muscles are resistant to acutemechanical injury.