Extended haplotype analysis of ovine ADRB3 using polymerase chain reaction single strand conformational polymorphism on two regions of the gene

The β3 adrenergic receptor (ADRB3) plays a critical role in the regulation of energy metabolism in mammals. In sheep, intronic polymorphism of the ADRB3 gene has been associated with lamb survival and various production traits. This study investigates variation in the ovine ADRB3 3' untranslated region (3'UTR), a region that may impact expression of the gene. Using PCR- single strand conformational polymorphism (SSCP), six unique patterns (named a-f) were observed in an approximately 304-bp amplicon. Sequencing revealed three single-nucleotide polymorphisms (c.*233A>C, c.*271G>C, c.*357A>T) and a single-nucleotide deletion (c.*257delG). Haplotype analyses showed that the previously described allele A defined by variation in the ovine ADRB3 intron can be divided into three haplotypes (Aa, Ab, and Ac). In total, 16 haplotypes through ovine ADRB3 were detected. This study suggests that ovine ADRB3 is highly polymorphic and that the extended haplotype analysis through the promoter, 5'UTR, coding sequence, intron, and 3'UTR needs to be performed to define the full extent of variation in this gene.     

The role of trimerization in the osmoregulated betaine transporter BetP

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.     

Nothing a hot bath won't cure: infection rates of amphibian chytrid fungus correlate negatively with water temperature under natural field settings

Dramatic declines and extinctions of amphibian populations throughout the world have been associated with chytridiomycosis, an infectious disease caused by the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd). Previous studies indicated that Bd prevalence correlates with cooler temperatures in the field, and laboratory experiments have demonstrated that Bd ceases growth at temperatures above 28°C. Here we investigate how small-scale variations in water temperature correlate with Bd prevalence in the wild. We sampled 221 amphibians, including 201 lowland leopard frogs (Rana [Lithobates] yavapaiensis), from 12 sites in Arizona, USA, and tested them for Bd. Amphibians were encountered in microhabitats that exhibited a wide range of water temperatures (10-50°C), including several geothermal water sources. There was a strong inverse correlation between the water temperature in which lowland leopard frogs were captured and Bd prevalence, even after taking into account the influence of year, season, and host size. In locations where Bd was known to be present, the prevalence of Bd infections dropped from 75-100% in water <15°C, to less than 10% in water >30°C. A strong inverse correlation between Bd infection status and water temperature was also observed within sites. Our findings suggest that microhabitats where water temperatures exceed 30°C provide lowland leopard frogs with significant protection from Bd, which could have important implications for disease dynamics, as well as management applications.There must be quite a few things a hot bath won't cure, but I don't know many of them--Sylvia Plath, "The Bell Jar" (1963).     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Whole-genome shotgun optical mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.     

Cyclodextrin-polyethylenimine conjugates for targeted in vitro gene delivery

Many human gene therapies will require cell-specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell-specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene-delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with beta-cyclodextrin on approximately 10% of the polymer's amines (termed CD-PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal-HI), which could anchor the protein to CD-PEI/DNA polyplexes. CD-PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4-fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal-HI to CD-PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal-HI. Because of the relative ease with which CD-binding moieties may be attached to various types of ligands, CD-PEI may be a generally useful material for testing novel cell-specific targeting compounds.     

Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, beta-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.