Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage

Effects of High-Molecular-Weight Dissolved Organic Matter on Nitrogen Dynamics in the Mississippi River Plume

The dynamics of N and its interactions with labile dissolved organic C (DOC), bacteria, and phytoplankton were studied to determine potential effects of dissolved organic matter (DOM) and light on N dynamics in surface waters of the Mississippi River (USA) plume in the Gulf of Mexico. Bacterial uptake of added labeled N compounds ( 15NH4+ or 15N-labeled dissolved free amino acids. DFAA) was stimulated more by high-molecular-weight (HMW, \u3el kDa) DOM than by low-molecular-weight (LMW, \u3c l kDa) DOM. An index that inversely indicated the presence of labile DOC was defined as the fraction of assimilated Amino acid-15N that was Recovered as 15N -Ammonium (ANRA), following the additions of high-levels (4 µM) of 15N -DFAA. ANRA ratios were high in the absence of other available carbon sources because heterotrophic bacteria were forced to use the added amino acids as a carbon source for respiration rather than as a nutrient source for biomass formation. In dynamic light/dark experiments, conducted with in situ populations of organisms, uptake rates of added 15NH4+ were significantly enhanced both by the presence of light and by the addition of HMW DOM. Uptake rates of added 15N -labeled DFAA were increased by the addition of HMW DOM but not by light. ANRA ratios were consistently lower in the presence of added HMW DOM than in controls. Added HMW DOM thus appeared to stimulate the incorporation of assimilated DFAA into bacterial biomass. Bacterial growth rates were relatively high in both light and dark bottles with DFAA additions and in light bottles with HMW DOM plus NH4+ additions, but they remained comparatively low in dark bottles with added NH4+ These results are consistent with the idea that bacterial N dynamics in these euphotic waters may be tightly coupled to photosynthetic activities over short time scales

Spectral stability of noncharacteristic isentropic Navier-Stokes boundary layers

Building on work of Barker, Humpherys, Lafitte, Rudd, and Zumbrun in the shock wave case, we study stability of compressive, or "shock-like", boundary layers of the isentropic compressible Navier-Stokes equations with gamma-law pressure by a combination of asymptotic ODE estimates and numerical Evans function computations. Our results indicate stability for gamma in the interval [1, 3] for all compressive boundary-layers, independent of amplitude, save for inflow layers in the characteristic limit (not treated). Expansive inflow boundary-layers have been shown to be stable for all amplitudes by Matsumura and Nishihara using energy estimates. Besides the parameter of amplitude appearing in the shock case, the boundary-layer case features an additional parameter measuring displacement of the background profile, which greatly complicates the resulting case structure. Moreover, inflow boundary layers turn out to have quite delicate stability in both large-displacement and large-amplitude limits, necessitating the additional use of a mod-two stability index studied earlier by Serre and Zumbrun in order to decide stability

A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia

Long-term survival still eludes most patients with leukemia and non-Hodgkin’s lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications

Stromal cell-derived factor and granulocyte-monocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy

ObjectiveIschemic heart failure is an increasingly prevalent global health concern with major morbidity and mortality. Currently, therapies are limited, and novel revascularization methods might have a role. This study examined enhancing endogenous myocardial revascularization by expanding bone marrow-derived endothelial progenitor cells with the marrow stimulant granulocyte-monocyte colony-stimulating factor and recruiting the endothelial progenitor cells with intramyocardial administration of the potent endothelial progenitor cell chemokine stromal cell-derived factor.MethodsIschemic cardiomyopathy was induced in Lewis rats (n = 40) through left anterior descending coronary artery ligation. After 3 weeks, animals were randomized into 4 groups: saline control, granulocyte-monocyte colony-stimulating factor only (GM-CSF only), stromal cell-derived factor only (SDF only), and combined stromal cell-derived factor/granulocyte-monocyte colony-stimulating factor (SDF/GM-CSF) (n = 10 each). After another 3 weeks, hearts were analyzed for endothelial progenitor cell density by endothelial progenitor cell marker colocalization immunohistochemistry, vasculogenesis by von Willebrand immunohistochemistry, ventricular geometry by hematoxylin-and-eosin microscopy, and in vivo myocardial function with an intracavitary pressure-volume conductance microcatheter.ResultsThe saline control, GM-CSF only, and SDF only groups were equivalent. Compared with the saline control group, animals in the SDF/GM-CSF group exhibited increased endothelial progenitor cell density (21.7 ± 3.2 vs 9.6 ± 3.1 CD34+/vascular endothelial growth factor receptor 2–positive cells per high-power field, P = .01). There was enhanced vascularity (44.1 ± 5.5 versus 23.8 ± 2.2 von Willebrand factor-positive vessels per high-power field, P = .007). SDF/GM-CSF group animals experienced less adverse ventricular remodeling, as manifested by less cavitary dilatation (9.8 ± 0.1 mm vs 10.1 ± 0.1 mm [control], P = .04) and increased border-zone wall thickness (1.78 ± 0.19 vs 1.41 ± 0.16 mm [control], P = .03). (SDF/GM-CSF group animals had improved cardiac function compared with animals in the saline control group (maximum pressure: 93.9 ± 3.2 vs 71.7 ± 3.1 mm Hg, P < .001; maximum dP/dt: 3513 ± 303 vs 2602 ± 201 mm Hg/s, P < .05; cardiac output: 21.3 ± 2.7 vs 13.3 ± 1.3 mL/min, P < .01; end-systolic pressure-volume relationship slope: 1.7 ± 0.4 vs 0.5 ± 0.2 mm Hg/μL, P < .01.)ConclusionThis novel revascularization strategy of bone marrow stimulation and intramyocardial delivery of the endothelial progenitor cell chemokine stromal cell-derived factor yielded significantly enhanced myocardial endothelial progenitor cell density, vasculogenesis, geometric preservation, and contractility in a model of ischemic cardiomyopathy