The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.     

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.     

Environmental and endogenous peroxisome proliferator-activated receptor gamma agonists induce bone marrow B cell growth arrest and apoptosis: interactions between mono(2-ethylhexyl)phthalate, 9-cis-retinoic acid, and 15-deoxy-Delta12,14-prostaglandin J2

The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARgamma agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), on developing B cells. MEHP inhibited [(3)H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor alpha ligand, 9-cis-retinoic acid, decreased [(3)H]thymidine incorporation synergistically, thereby implicating activation of a PPARgamma-retinoid X receptor alpha complex. These results were similar to those obtained with the natural PPARgamma ligand 15d-PGJ(2). At moderate MEHP concentrations (25 or 100 microM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [(3)H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ(2) and MEHP significantly enhanced the inhibition of [(3)H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ(2)-induced death does not result from a decrease in NF-kappaB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ(2), to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARgamma activation.     

Long-term effects of sterol depletion in C. elegans: sterol content of synchronized wild-type and mutant populations

Three major long-term effects of sterol deprivation in Caenorhabditis elegans are described. 1) The life expectancy of sterol-deprived wild-type animals is decreased by more than 40%. Similar decreases are found in animals carrying mutations in the daf-9, daf-12, daf-16, and clk-1 genes, suggesting that previously described aging pathways involving these genes are not involved in the life-extending effects of sterols. 2) There is a premature loss of motility, measured by response to mild touch. 3) There is a rapid postreproductive onset of sarcopenia (muscle wasting) as measured by total body fluorescence in a myo3::GFP-expressing strain. We also report that five sterols (the desmethylsterols cholesterol, 7-dehydrocholesterol, and lathosterol and the 4alpha-methyl sterols lophenol and 4alpha-methyl-cholesta-Delta8(14)-en-3beta-ol) are found in significant amounts at all stages of development and aging in cholesterol-fed animals. Supplying any one of these as the sole sterol confers similar protection from the long-term effects of sterol deprivation. These findings suggest that sterols are required continuously throughout the animal's life.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Novel role for decay-accelerating factor in coxsackievirus A21-mediated cell infectivity

Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses. Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21 (CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15 min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous or induced ICAM-1.     

Sources of variation in growth, form, and survival in dwarf and normal-stature pitch pines (Pinus rigida, Pinaceae) in long-term transplant experiments

Determining the relative contributions of genetic and environmental factors to phenotypic variation is critical for understanding the evolutionary ecology of plant species, but few studies have examined the sources of phenotypic differentiation between nearby populations of woody plants. We conducted reciprocal transplant experiments to examine sources of variation in growth rate, form, survival, and maturation in a globally rare dwarf population of pitch pine (Pinus rigida) and in surrounding populations of normal-stature pitch pines on Long Island, New York. Transplants were monitored over a 6-yr period. The influence of seedling origin on height, growth rate, survival, and form (single-stemmed vs. multi-stemmed growth habit) was much smaller than the effect of transplanting location. Both planting site and seed origin were important factors in determining time to reproduction; seedlings originating from dwarf populations and seedlings planted at the normal-stature site reproduced earliest. These results suggest that many of the differences between dwarf and normal-stature pitch pines may be due more to plastic responses to environmental factors than to genetic differentiation among populations. Therefore, preservation of the dwarf pine habitat is essential for preserving dwarf pine communities; the dwarf pines cannot be preserved ex situ.     

Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent

The recruitment of polymorphonuclear leukocytes (PMN) from the vascular space to the alveolar air space is an early event in host defense against pneumococcal pneumonia. Pneumolysin is a virulence factor for Streptococcus pneumoniae, but a specific role for pneumolysin in neutrophil-endothelial cell interactions has not been investigated. Using a Transwell system, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to wild-type S. pneumoniae (D39) and a pneumolysin-deficient mutant (plnA(-)) incubated on the abluminal surface of the monolayer. S. pneumoniae induction of PMN migration was dose dependent and elicited by > or =10(5) D39. Mutants lacking pneumolysin had dramatically reduced potency for eliciting PMN migration compared with the parent strain (5 x 10(6) plnA(-) elicits 18.6% PMN migration vs. 55.5% for 5 x 10(6) D39). The disparity between D39 and plnA(-) persisted in ethanol-fixed bacteria, consistent with the properties of pneumolysin. Neither conditioned medium from D39 nor purified pneumolysin elicited PMN migration to the same extent as the intact D39, suggesting that the role of pneumolysin in eliciting PMN migration requires a more complex interaction between the organism, the endothelium, and the PMN. Both D39 and plnA(-) adhered to, and translocated across, the endothelium in the abluminal to luminal direction and elicited similar levels of IL-8 production. Neither strain elicited upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin, and they did not cause translocation of NF-kappaB to the nucleus. These findings demonstrate a novel role for pneumolysin in pneumococcus-induced PMN recruitment across the pulmonary endothelium.     

Effect of muscle wrapping on model estimates of neck muscle strength

Of the computational models of the cervical spine reported in the literature, not one takes into account the changes in muscle paths due to the underlying vertebrae. Instead, all model the individual muscle paths as straight-line segments. The major aim of this study was to quantify the changes in muscle moment arm, muscle force and joint moment due to muscle wrapping in the cervical spine. Five muscles in a straight-line model of the cervical spine were wrapped around underlying vertebrae, and the results obtained from this model were compared against the original. The two models were then validated against experimental and computational data. Results show that muscle wrapping has a significant effect on muscle moment arms and therefore joint moments and should not be neglected.