Isolation and characterization of 12 polymorphic microsatellite markers in the tropical house gecko (Hemidactylus mabouia)

We present 12 variable microsatellite loci isolated from the invasive tropical house gecko, Hemidactylus mabouia. Polymerase chain reaction (PCR) primers were tested on 39 individuals from two locations in Miami, Florida. Heterozygote deficiency was detected for four loci, and we attribute this to possible null alleles or population substructure. Some loci successfully amplified PCR products in several congeners, indicating their potential for use in other geckos.     

Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) and Nuclear Factor-κB (NF-κB): A FEED-FORWARD LOOP FOR SYSTEMIC AND VASCULAR INFLAMMATION*

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.     

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

Background

HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.

Augmented sympathetic tone alters muscle metabolism with exercise: lack of evidence for functional sympatholysis

Shoemaker, J. Kevin, Prasant Pandey, Michael D. Herr, DavidH. Silber, Qing X. Yang, Michael B. Smith, Kristen Gray, and LawrenceI. Sinoway. Augmented sympathetic tone alters muscle metabolismwith exercise: lack of evidence for functional sympatholysis. J. Appl. Physiol. 82(6):1932-1938, 1997.It is unclear whether sympathetic tone opposesdilator influences in exercising skeletal muscle. We examined highlevels of sympathetic tone, evoked by lower body negative pressure(LBNP, 60 mmHg) on intramuscular pH and phosphocreatine (PCr)levels (³¹P-nuclear magnetic resonance spectroscopy) duringgraded rhythmic handgrip (30 contractions/min; ~17, 34, 52 and 69%maximal voluntary contraction). Exercise was performedwith LBNP and without LBNP (Control). At the end of exercise, LBNPcaused lower levels of muscle pH (6.59 ± 0.09) comparedwith Control (6.78 ± 0.05; P < 0.05). PCr recovery, an index of mitochondrial respiration, was lessduring the recovery phase of the LBNP trial. Exercise mean arterialpressure was not altered by LBNP. The protocols were repeated withmeasurements of forearm blood flow velocity and deep venous samples(active forearm) of hemoglobin (Hb) saturation, pH, and lactate. WithLBNP, mean blood velocity was reduced at rest, during exercise, andduring recovery compared with Control (P < 0.05). Also, venous Hbsaturation and pH levels during exercise and recovery were lower withLBNP and lactate was higher compared with Control(P < 0.05). We concludethat LBNP enhanced sympathetic tone and reduced oxygen transport. Athigh workloads, there was a greater reliance on nonoxidativemetabolism. In other words, sympatholysis did not occur.

     

Molecular vehicles for targeted drug delivery

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.     

Ecological genomics predicts climate vulnerability in an endangered southwestern songbird

Few regions have been more severely impacted by climate change in the USA than the Desert Southwest. Here, we use ecological genomics to assess the potential for adaptation to rising global temperatures in a widespread songbird, the willow flycatcher (Empidonax traillii), and find the endangered desert southwestern subspecies (E. t. extimus) most vulnerable to future climate change. Highly significant correlations between present abundance and estimates of genomic vulnerability – the mismatch between current and predicted future genotype–environment relationships – indicate small, fragmented populations of the southwestern willow flycatcher will have to adapt most to keep pace with climate change. Links between climate‐associated genotypes and genes important to thermal tolerance in birds provide a potential mechanism for adaptation to temperature extremes. Our results demonstrate that the incorporation of genotype–environment relationships into landscape‐scale models of climate vulnerability can facilitate more precise predictions of climate impacts and help guide conservation in threatened and endangered groups.     

Muscle fiber structure in an aging long-lived seabird,the black-legged kittiwake (Rissa tridactyla)

Many long-lived animals do not appear to show classic signs of aging, perhaps because they show negligible senescence until dying from “catastrophic” mortality. Muscle senescence is seldom examined in wild animals, yet decline in muscle function is one of the first signs of aging in many lab animals and humans. Seabirds are an excellent study system for physiological implications of aging because they are long-lived animals that actively forage and reproduce in the wild. Here, we examined linkages between pectoralis muscle fiber structure and age in black-legged kittiwakes (Rissa tridactyla). Pectoralis muscle is the largest organ complex in birds, and responsible for flight and shivering. We obtained and fixed biopsies from wild black-legged kittiwakes of known age. We then measured muscle fiber diameter, myonuclear domain and capillaries per fiber area among birds of differing ages. All muscle parameters were independent of age. Number of nuclei per mm of fiber showed a positive correlation with muscle fiber cross-sectional area, and myonuclear domain increased with muscle fiber diameter. Thus, as muscle fibers increased in size, they may not have recruited satellite cells, increasing the protein turnover load per nuclei. We conclude that senescence in a long-lived bird with an active lifestyle, does not entail mammalian-like changes in muscle structure.     

Intrinsic site‐selectivity of ubiquitin dimer formation

The post‐translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme‐catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme‐free system by measuring the rate constants for thiol–disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles.     

Evidence for the role of hindbrain orexin-1 receptors in the control of meal size

Hypothalamic orexin neurons project to the hindbrain, and 4th-ventricle intracerebroventricular (4th-icv) injection of orexin-A treatment increases food intake. We assessed the effects of hindbrain orexin-A and the orexin-1-receptor antagonist SB334867 on meal pattern in rats consuming standard chow. When injected 4th-icv shortly before dark onset, lower doses of orexin-A increased food intake over a 2-h period by increasing the size of the first meal relative to vehicle, whereas the highest dose increased food intake by causing the second meal to be taken sooner. Conversely, hindbrain SB334867 reduced food intake by decreasing the size of the first meal of the dark phase. We also examined the effects of 4th-icv orexin-A and SB334867 on locomotor activity. Only the highest dose of orexin-A increased activity, and SB334867 had no effect. In addition, hindbrain SB334867 induced c-Fos in the nucleus of the solitary tract. These data support the suggestion that endogenous hindbrain orexin-A acts to limit satiation. Both orexin-A and the pancreatic satiation hormone amylin require an intact area postrema to affect food intake, so we asked whether 4th-icv orexin-A impairs the satiating effect of peripheral amylin treatment. Amylin reduced the size of the first meal of the dark cycle when rats were pretreated with 4th-icv saline, yet amylin was ineffective after 4th-icv orexin-A pretreatment. Using double-label immunohistochemistry, we determined that some orexin-A fibers in the area postrema are located in proximity to amylin-responsive neurons. Therefore, hindbrain orexin-A may increase food intake, in part, by reducing the ability of rats to respond to amylin during a meal.