Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.     

Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice

Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.     

Ept7 influences estrogen action in the pituitary gland and body weight of rats

Estrogens control many aspects of pituitary gland biology, including regulation of lactotroph homeostasis and synthesis and secretion of prolactin. In rat models, these actions are strain specific and heritable, and multiple quantitative trait loci (QTL) have been mapped that impact the responsiveness of the lactotroph to estrogens. One such QTL, Ept7, was mapped to RNO7 in female progeny generated in an intercross between BN rats, in which the lactotroph population is insensitive to estrogens, and ACI rats, which develop lactotroph hyperplasia/adenoma and associated hyperprolactinemia in response to estrogen treatment. The primary objective of this study was to confirm the existence of Ept7 and to quantify the impact of this QTL on responsiveness of the pituitary gland of female and male rats to 17β-estradiol (E2) and diethylstilbestrol (DES), respectively. Secondary objectives were to determine if Ept7 influences the responsiveness of the male reproductive tract to DES and to identify other discernible phenotypes influenced by Ept7. To achieve these objectives, a congenic rat strain that harbors BN alleles across the Ept7 interval on the genetic background of the ACI strain was generated and characterized to define the effect of administered estrogens on the anterior pituitary gland and male reproductive tissues. Data presented herein indicate Ept7 exerts a marked effect on development of lactotroph hyperplasia in response to estrogen treatment, but does not affect atrophy of the male reproductive tissues in response to hormone treatment. Ept7 was also observed to exert gender specific effects on body weight in young adult rats.     

Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks

MicroRNAs (miRNAs) are key regulators of gene expression in development and stress responses in most eukaryotes. We globally profiled plant miRNAs in response to infection of bacterial pathogen Pseudomonas syringae pv. tomato (Pst). We sequenced 13 small-RNA libraries constructed from Arabidopsis at 6 and 14 h post infection of non-pathogenic, virulent and avirulent strains of Pst. We identified 15, 27 and 20 miRNA families being differentially expressed upon Pst DC3000 hrcC, Pst DC3000 EV and Pst DC3000 avrRpt2 infections, respectively. In particular, a group of bacteria-regulated miRNAs targets protein-coding genes that are involved in plant hormone biosynthesis and signaling pathways, including those in auxin, abscisic acid, and jasmonic acid pathways. Our results suggest important roles of miRNAs in plant defense signaling by regulating and fine-tuning multiple plant hormone pathways. In addition, we compared the results from sequencing-based profiling of a small set of miRNAs with the results from small RNA Northern blot and that from miRNA quantitative RT-PCR. Our results showed that although the deep-sequencing profiling results are highly reproducible across technical and biological replicates, the results from deep sequencing may not always be consistent with the results from Northern blot or miRNA quantitative RT-PCR. We discussed the procedural differences between these techniques that may cause the inconsistency.     

Macroborer presence on corals increases with nutrient input and promotes parrotfish bioerosion

Coral Reefs - Bioerosion by reef-dwelling organisms influences net carbonate budgets on reefs worldwide. External bioeroders, such as parrotfish and sea urchins, and internal bioeroders, including...     

Evidence that the protein tyrosine phosphatase (PC12,Br7,Sl) gamma (-) isoform modulates chondrogenic patterning and growth

One of the earliest events in bone morphogenesis is the condensation of embryonic mesenchymal cells into chondroblasts and their subsequent proliferation and differentiation into chondrocytes. During this time, certain signaling cascades operate to establish proper patterning and differentiation of the cartilaginous skeleton. Characterization of the signaling pathways involved in these processes remains to be accomplished. We have identified a novel murine cytosolic tyrosine phosphatase termed PTPPBS gamma (+/-) which is a member of the PTP PC12,Br7,Sl (PTPPBS) family. Spatio-temporal expression analysis of the members of this tyrosine phosphatase family demonstrates significant expression of the gamma (-) splice variant in the cartilaginous skeleton. Using an embryonic mandibular explant culture system to serve as a model for cartilage formation, we examined the potential roles of the PTPPBS gamma phosphatase by loss-of-function studies achieved with antisense oligodeoxynucleotides. These studies demonstrated that loss of expression of the PTPPBS gamma (-) isoform resulted in abnormal patterning of Meckel's cartilage and an increase in the size of the chondrogenic regions. In gamma antisense-treated explants, bromodeoxyuridine-pulse labeling studies revealed increased proliferation of chondroblasts bordering along precartilaginous condensations and bordering populations of maturing chondrocytes. These studies provide evidence that in early skeletal development, PTPPBS gamma may regulate the rate of chondroblast proliferation in the cartilaginous skeleton.     

Enzymatic incorporation of a third nucleobase pair

DNA polymerases are identified that copy a non-standard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard 'small' donor-donor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo-) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.     

Identification and Validation of a Potent Dual Inhibitor of the P. falciparum M1 and M17 Aminopeptidases Using Virtual Screening

The Plasmodium falciparum PfA-M1 and PfA-M17 metalloaminopeptidases are validated drug targets for the discovery of antimalarial agents. In order to identify dual inhibitors of both proteins, we developed a hierarchical virtual screening approach, followed by in vitro evaluation of the highest scoring hits. Starting from the ZINC database of purchasable compounds, sequential 3D-pharmacophore and molecular docking steps were applied to filter the virtual ‘hits’. At the end of virtual screening, 12 compounds were chosen and tested against the in vitro aminopeptidase activity of both PfA-M1 and PfA-M17. Two molecules showed significant inhibitory activity (low micromolar/nanomolar range) against both proteins. Finally, the crystal structure of the most potent compound in complex with both PfA-M1 and PfA-M17 was solved, revealing the binding mode and validating our computational approach.     

A metal ion–dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.