Calculation of higher protonation states and of a new resting state for vanadium chloroperoxidase using QM/MM, with an Atom-in-Molecules analysis.

Earlier QM/MM studies of the resting state of vanadium chloroperoxidase (VCPO) focused on the diprotonated states of the vanadate cofactor. Herein, we report a new extensive QM/MM study that includes the tri- and quadprotonated states of VCPO at neutral pH. We identify certain di- and triprotonated states as being candidates for the resting state based on a comparison of relative energies. The quadprotonated states as well as some of the triprotonated states are ruled out as the resting state. An Atoms-in-Molecules (AIM) analysis of the complex hydrogen bonding around the vanadate cofactor helps to explain the relative energies of the protonation states considered herein, and it also indicates new hydrogen bonding which has not been recognized previously. A Natural Bond Orbital (NBO) study is presented to give a better understanding of the electronic structure of the vanadate co-factor.

Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics.

BACKGROUND: Vascular patterning depends on coordinated timing of arteriovenous specification of endothelial cells and the concomitant hemodynamic forces supplied by the onset of cardiac function. Using a combination of 3D imaging by OPT and embryo registration techniques, we sought to identify structural differences between three different mouse models of cardiovascular perturbation. RESULTS: Endoglin mutant mice shared a high degree of similarity to Mlc2a mutant mice, which have been shown to have a primary developmental heart defect causing secondary vessel remodeling failures. Dll4 mutant mice, which have well-characterized arterial blood vessel specification defects, showed distinct differences in vascular patterning when compared to the disruptions seen in Mlc2a-/- and Eng-/- models. While Mlc2a-/- and Eng-/- embryos exhibited significantly larger atria than wild-type, Dll4-/- embryos had significantly smaller hearts than wild-type, but this quantitative volume decrease was not limited to the developing atrium. Dll4-/- embryos also had atretic dorsal aortae and smaller trunks, suggesting that the cardiac abnormalities were secondary to primary arterial blood vessel specification defects. CONCLUSIONS: The similarities in Eng-/- and Mlc2a-/- embryos suggest that Eng-/- mice may suffer from a primary heart developmental defect and secondary defects in vessel patterning, while defects in Dll4-/- embryos are consistent with primary defects in vessel patterning.

3D imaging, registration, and analysis of the early mouse embryonic vasculature.

BACKGROUND: Cardiovascular development requires the input of a large number of molecular signaling molecules, and undergoes tightly regulated, three-dimensional developmental patterning. Conventional developmental biology techniques have successfully identified many of the signaling cascades and molecular cues necessary for proper cardiovascular development, which has furnished us with a wealth of biochemical, molecular, and biologically functional information on how tightly linked cardiac and vascular development are. Still missing, however, is a genuine appreciation of the three-dimensional (3D) nature of these important developmental steps. RESULTS: Optical projection tomography (OPT) is a 3D imaging technique that allows for high-resolution imaging of early mouse embryos and their developing cardiovascular systems when a PECAM-1 antibody stain is used to highlight the vascular branching. Reported here is a method in which several 3D images of mouse embryo vasculatures can be registered, thus allowing for analysis of within-strain variance between genetically identical mouse pups. Post-registration, small differences in somitogenesis and ventricular trabeculation patterning can be visualized in mouse pups that differ by as little as a few hours of gestational time. Additionally, similarity metrics (cross-correlation values) can be calculated to quantify similarities and differences. Two different mouse strains are analyzed (C57Bl/6 and CD-1), and similar results are recognized in each strain. CONCLUSIONS: Visualizing the cardiovascular system in such a precise 3D manner allows for more accuracy in describing the steps that take place during cardiovascular development. This novel method will be applicable to many developmental biology questions in other organ systems and other species.

Hedgehog regulates distinct vascular patterning events through VEGF-dependent and -independent mechanisms.

Despite the clear importance of Hedgehog (Hh) signaling in blood vascular development as shown by genetic analysis, its mechanism of action is still uncertain. To better understand the role of Hh in vascular development, we further characterized its roles in vascular development in mouse embryos and examined its interaction with vascular endothelial growth factor (VEGF), a well-known signaling pathway essential to blood vascular development. We found that VEGF expression in the mouse embryo depended on Hh signaling, and by using genetic rescue approaches, we demonstrated that the role of Hh both in endothelial tube formation and Notch-dependent arterial identity was solely dependent on its regulation of VEGF. In contrast, overactivation of the Hh pathway through deletion of Patched1 (Ptch1), a negative regulator of Hh signaling, resulted in reduced vascular density and increased Delta-like ligand 4 expression. The Ptch1 phenotype was independent of VEGF pathway dysregulation and was not rescued when Delta-like ligand 4 levels were restored to normal. These findings establish that Hh uses both VEGF- and Notch-dependent and -independent mechanisms to pattern specific events in early blood vascular development.

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo.

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

Prostatic extramedullary leukemia as a first site of relapse of acute nonlymphocytic leukemia.

Extramedullary leukemia (EML) is an uncommon clinical diagnosis in patients with acute nonlymphocytic leukemia (ANLL). Prostatic EML as a first site of ANLL relapse is even more rare. To our knowledge, only three cases have been reported. We describe an additional case of prostatic EML as a site of ANLL relapse. An asymptomatic male in ANLL remission was found to have a normal prostate-specific antigen (PSA) and a myeloid leukemic infiltrate in a newly diagnosed prostate nodule. Staging was negative for ANLL relapse. Local prostate radiation resolved the nodule, and the post-treatment needle biopsies were negative. He subsequently developed clinical relapse in bone marrow, blood, and small bowel and received salvage chemotherapy with mitoxantrone and etoposide.