ABSTRACT
In this study, we present a new approach to variable number tandem repeats (VNTR) analysis using the QIAxcel capillary electrophoresis system and a software-integrated peak calling function. Allelic ladders representing 15 mycobacterial interspersed repetitive units (MIRU)-VNTR loci were used to define peak calling tables thereby enabling high precision Mycobacterium tuberculosis strain identification.
Subject(s)
Automation, Laboratory/methods , DNA, Bacterial/genetics , Minisatellite Repeats , Molecular Typing/methods , Mycobacterium tuberculosis/genetics , Electrophoresis, Capillary/methods , Mycobacterium tuberculosis/classificationABSTRACT
Therapeutic angiogenesis is a promising strategy for treating ischemia. The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration and tube formation, and plays the critical role in developmental angiogenesis. We developed poly(lactic-co-glycolic-acid) (PLGA)-based S1P-containing microparticles (PLGA-S1P), which are biodegradable and continuously release S1P, and studied the effects of PLGA-S1P on neovascularization in murine ischemic hindlimbs. Intramuscular injections of PLGA-S1P stimulated blood flow in C57BL/6 mice dose-dependently, with repeated administrations at a 3-day interval, rather than a single bolus or 6-day interval, over 28 days conferring the optimal stimulating effect. In Balb/c mice that exhibit limb necrosis and dysfunction due to retarded blood flow recovery, injections of PLGA-S1P stimulated blood flow with alleviation of limb necrosis and dysfunction. PLGA-S1P alone did not induce edema in ischemic limbs, and rather blocked vascular endothelial growth factor-induced edema. PLGA-S1P not only increased the microvessel densities in ischemic muscle, but promoted coverage of vessels with smooth muscle cells and pericytes, thus stabilizing vessels. PLGA-S1P stimulated Akt and ERK with increased phosphorylation of endothelial nitric oxide synthase in ischemic muscle. The effects of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methylester, showed that PLGA-S1P-induced blood flow stimulation was partially dependent on nitric oxide. Injections of PLGA-S1P also increased the expression of angiogenic factors and the recruitment of CD45-, CD11b- and Gr-1-positive myeloid cells, which are implicated in post-ischemic angiogenesis, into ischemic muscle. These results indicate that PLGA-based, sustained local delivery of S1P is a potentially useful therapeutic modality for stimulating post-ischemic angiogenesis.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/physiology , Ischemia/drug therapy , Ischemia/physiopathology , Lactic Acid/administration & dosage , Lysophospholipids/administration & dosage , Neovascularization, Physiologic/drug effects , Nitric Oxide Synthase Type III/physiology , Polyglycolic Acid/administration & dosage , Proto-Oncogene Proteins c-akt/physiology , Sphingosine/analogs & derivatives , Animals , Delayed-Action Preparations/administration & dosage , Disease Models, Animal , Hindlimb/blood supply , Hindlimb/drug effects , Ischemia/enzymology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microspheres , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Polylactic Acid-Polyglycolic Acid Copolymer , Random Allocation , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Sphingosine/administration & dosageABSTRACT
To understand the mechanism of the very slow block to polyspermy in physiologically polyspermic eggs of the newt Cynops pyrrhogaster, we used confocal laser microscopy to determine the distribution of gamma-tubulin and cyclin B1 in fertilized eggs. More gamma-tubulin was localized in the animal hemisphere than in the vegetal. The centrosomes of the principal sperm nucleus and the zygote nucleus had much accumulated gamma-tubulin, but little gamma-tubulin was associated with the centrosomes of the accessory sperm nuclei. These results are consistent with observations that the largest sperm aster is associated with the principal sperm nucleus. More cyclin B1 appeared in the animal hemisphere than in the vegetal at the end of interphase. The zygote nucleus had much accumulated cyclin B1, but little cyclin B1 was associated with the accessory sperm nuclei. Cyclin B1 disappeared earlier around the zygote nucleus at metaphase than around the accessory sperm nuclei. These findings correspond well with the earlier entry and exit into metaphase in the zygote nucleus than in the accessory sperm nuclei in newt eggs, supporting our maturation-promoting factor (MPF) model that accounts for the mechanism of nuclear degeneration in physiologically polyspermic eggs. Cyclin B1 began to accumulate in the nucleus during interphase in synchronous cleavage, and its greatest expression was in the centrosomes and the nucleus at prometaphase.
