Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.     

Photolabeling study of the ligand binding domain of natriuretic peptide receptor A: development of a model

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are loop-shaped peptidic hormones that have multiple actions on body fluid homeostasis. Their physiological effects are mediated through the activation of their receptor, natriuretic peptide receptor A (NPRA). This receptor is a member of the membrane guanylyl cyclase family and catalyzes cyclic guanosine monophosphate (cGMP) production following its activation. To map the binding site of human NPRA, we applied the methionine proximity assay method to this receptor. We photolabeled NPRA mutants, presenting a single methionine in the binding domain of the receptor, and used benzoylphenylalanine- (Bpa-) substituted peptides at positions 0, 3, 18, 26, and 28 of the ligand. We identified that the N-terminus of the peptide is interacting with the region between Asp(177) and Val(183) of the receptor. Arg(3) is interacting in the vicinity of Phe(172). Leu(18) binds close to Val(116). Phe(26) binds in the vicinity of His(195), and the C-terminal Tyr(28) is located close to Met(173). We next proceeded with photolabeling of a dual Bpa-substituted peptide and showed that the N-terminus and Leu(18) interact with opposite receptor subunits. On the basis of our results, a molecular model of peptide-bound NPRA was developed by homology modeling with the C-type natriuretic peptide- (CNP-) bound natriuretic peptide receptor C (NPRC) crystal structure. The model has been validated by molecular dynamics simulations. Our work provides a rational basis for interpreting and predicting natriuretic peptide binding to the human NPRA.     

Inhibition of endothelial cell movement and tubulogenesis by human recombinant soluble melanotransferrin: involvement of the u-PAR/LRP plasminolytic system

We have previously demonstrated that human recombinant soluble melanotransferrin (hr-sMTf) interacts with the single-chain zymogen pro urokinase-type plasminogen activator (scu-PA) and plasminogen. In the present work, the impact of exogenous hr-sMTf on endothelial cells (EC) migration and morphogenic differentiation into capillary-like structures (tubulogenesis) was assessed. hr-sMTF at 10 nM inhibited by 50% the migration and tubulogenesis of human microvessel EC (HMEC-1). In addition, in hr-sMTf-treated HMEC-1, the expression of both urokinase-type plasminogen activator receptor (u-PAR) and low-density lipoprotein receptor-related protein (LRP) are down-regulated. However, fluorescence-activated cell sorting analysis revealed a 25% increase in cell surface u-PAR in hr-sMTf-treated HMEC-1, whereas the binding of the urokinase-type plasminogen activator (u-PA)*plasminogen activator inhibitor-1 (PAI-1) complex is decreased. This reduced u-PA-PAI-1 binding is correlated with a strong inhibition of the HMEC-1 plasminolytic activity, indicating that exogenous hr-sMTf treatment alters the internalization and recycling processes of free and active u-PAR at the cellular surface. Overall, these results demonstrate that exogenous hr-sMTf affects plasminogen activation at the cell surface, thus leading to the inhibition of EC movement and tubulogenesis. These results are the first to consider the potential use of hr-sMTf as a possible therapeutic agent in angiogenesis-related pathologies.     

Mitotic recombination and genetic changes in Saccharomyces cerevisiae during wine fermentation

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 x 10(-5) to 3 x 10(-5) per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.     

The Population Decline and Extinction of Darwin’s Frogs

Darwin’s frogs (Rhinoderma darwinii and R. rufum) are two species of mouth-brooding frogs from Chile and Argentina. Here, we present evidence on the extent of declines, current distribution and conservation status of Rhinoderma spp.; including information on abundance, habitat and threats to extant Darwin’s frog populations. All known archived Rhinoderma specimens were examined in museums in North America, Europe and South America. Extensive surveys were carried out throughout the historical ranges of R. rufum and R. darwinii from 2008 to 2012. Literature review and location data of 2,244 archived specimens were used to develop historical distribution maps for Rhinoderma spp. Based on records of sightings, optimal linear estimation was used to estimate whether R. rufum can be considered extinct. No extant R. rufum was found and our modelling inferred that this species became extinct in 1982 (95% CI, 1980–2000). Rhinoderma darwinii was found in 36 sites. All populations were within native forest and abundance was highest in Chiloé Island, when compared with Coast, Andes and South populations. Estimated population size and density (five populations) averaged 33.2 frogs/population (range, 10.2–56.3) and 14.9 frogs/100 m² (range, 5.3–74.1), respectively. Our results provide further evidence that R. rufum is extinct and indicate that R. darwinii has declined to a much greater degree than previously recognised. Although this species can still be found across a large part of its historical range, remaining populations are small and severely fragmented. Conservation efforts for R. darwinii should be stepped up and the species re-classified as Endangered.     

The placenta theory and the origin of infantile hemangioma

The pathogenesis of infantile hemangioma is unknown. In recent years, much of the focus has been placed at identifying the cell type(s) responsible for tumor initiation. New discoveries in infantile hemangioma suggest an involvement of progenitor cells in the pathogenesis of this vascular tumor. Both embryonic and extra-embryonic tissues have been postulated as potential sources for these progenitor cells. This review focuses on the placental theory, which proposes that a fetal placental progenitor is the cell type of origin for infantile hemangioma. Special emphasis will be placed on placental vasculogenesis and the presence and transit of placental progenitor cells during gestation.     

Serum hemorphin‐7 levels are decreased in obesity

Objective:

Hemorphin peptides exhibit biological activities that interfere with the endorphin system, the inflammatory response, and blood‐pressure control. VV‐hemorphin‐7 and LVV‐hemorphin‐7 peptides exert a hypotensive effect, in particular, by inhibiting the renin–angiotensin system. Furthermore, levels of circulating hemorphin‐7 peptides have been found to be decreased in diseases such as type 1 and type 2 diabetes.

Design and Methods:

Because type 2 diabetes and obesity share common features, such as insulin resistance, microinflammation, high glomerular‐filtration rate (GFR), and cardiovascular risk, we evaluated serum VV‐hemorphin‐7 like immunoreactivity (VVH7‐i.r.) levels, using an enzyme‐linked immunosorbent assay method, on a group of 54 obese subjects without diabetes or hypertension, compared with a group of 33 healthy normal‐weight subjects.

Results:

Circulating VVH7‐i.r. levels were significantly decreased in the obese group compared with the control group (1.98 ± 0.19 vs. 4.86 ± 0.54 µmol/l, respectively, P < 0.01), and a significant negative correlation between VVH7‐i.r. and diastolic blood pressure (DBP) was found in obese patients (r = ?0.35, P = 0.011). There was no significant correlation between VVH7‐i.r. level and insulin resistance, metabolic syndrome, or GFR.

Conclusions:

The decreased serum hemorphin‐7 found in obese subjects, as in diabetes, may contribute to the development of hypertension and to the cardiovascular risk associated with these metabolic diseases.

     

Hydroxamates as substrates and inhibitors for FMN-dependent 2-hydroxy acid dehydrogenases

Long-chain hydroxy acid oxydase (HAO) is a member of a flavoenzyme family with significant amino acid sequence similarity and strongly conserved three-dimensional structure; in particular, active-site amino acids involved in catalysis are invariant, with one exception, and numerous enzymatic studies suggest an identical chemical mechanism involving an intermediate carbanion for all family members. Known physiological substrates are a variety of L-2-hydroxy acids. Peroxisomal HAO differs from the other family members in that its actual physiological substrate is not known; it was first described as an L-amino acid oxidase, and recently was identified as an enzyme that converts creatol (hydroxycreatinine) to methylguanidine (a metabolite involved in a variety of uremic syndromes). Creatol (2-amino-5-hydroxy-1-methyl-4(5H)imidazolone) is not a 2-hydroxy acid. We show in this work that 2-hydroxyphenyl acetohydroxamate (HYPAH, the hydroxamate of mandelic acid), a compound that bears similarity both to mandelate (one of the best substrates known) and to creatol, is turned over by HAO, but between 10- and 100-fold less efficiently than mandelate itself. The compound also binds to the active site of homologous flavocytochrome b(2) (L-lactate dehydrogenase). Comparative pH-rate studies for mandelate and its hydroxamate suggest that HYPAH may bind in its ionized form. Both pH-rate profiles are bell-shaped curves, as are those determined for two other family members, flavocytochrome b(2) and mandelate dehydrogenase; while the group with an acid pK(a) between 5 and 6 is most likely the active-site histidine (the residue which abstracts the substrate C2 proton), the identity of the basic group is less clear. It has been proposed to be one of the active site arginines (Lehoux, I., and Mitra, B. (1999) Biochemistry38, 5836-5848); we suggest as an alternative that it could be the lysine residue that interacts with the flavin N1 and O2 positions and stabilizes the negative charge of reduced flavin. In addition to these studies, we have found that HAO is competitively inhibited by benzohydroxamate, which is one atom shorter than HYPAH; its affinity is nearly 100-fold lower than that of the substrate, in contrast to the strong inhibition it exerts on mandelate racemase (Maurice, St. M., and Bearne, S. L. (2000) Biochemistry39, 13324-13335). In the latter case, the 100-fold higher affinity compared to mandelate was proposed to arise from the fact that the hydroxamate can mimic the enolic intermediate which lies on the reaction pathway after C2 proton abstraction. Thus our results do not support the existence of a similar enolic intermediate for HAO (and probably its homologues), although they do not disprove it.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.