A cell-permeable biscyclooctyne as a novel probe for the identification of protein sulfenic acids

Reactive oxygen species act as important second messengers in cell signaling and homeostasis through the oxidation of protein thiols. However, the dynamic nature of protein oxidation and the lack of sensitivity of existing molecular probes have hindered our understanding of such reactions; therefore, new tools are required to address these challenges. We designed a bifunctional variant of the strained bicyclo[6.1.0]nonyne (BCN-E-BCN) that enables the tagging of intracellular protein sulfenic acids for biorthogonal copper-free click chemistry. In validation studies, BCN-E-BCN binds the sulfenylated form of the actin-severing protein cofilin, while mutation of the cognate cysteine residues abrogates its binding. BCN-E-BCN is cell permeable and reacts rapidly with cysteine sulfenic acids in cultured cells. Using different azide-tagged conjugates, we demonstrate that BCN-E-BCN can be used in various applications for the detection of sulfenylated proteins. Remarkably, cycloaddition of an azide-tagged fluorophore to BCN-E-BCN labelled proteins produced in vivo can be visualized by fluorescence microscopy to reveal their subcellular localization. These findings demonstrate a novel and multifaceted approach to the detection and trapping of sulfenic acids

WILLINGNESS-TO-PAY FOR VALUE ADDED BRED HEIFER CHARACTERISTICS

Livestock Production/Industries,

PON1 status does not influence cholinesterase activity in Egyptian agricultural workers exposed to chlorpyrifos.

Animal studies have shown that paraoxonase 1 (PON1) genotype can influence susceptibility to the organophosphorus pesticide chlorpyrifos (CPF). However, Monte Carlo analysis suggests that PON1 genotype may not affect CPF-related toxicity at low exposure conditions in humans. The current study sought to determine the influence of PON1 genotype on the activity of blood cholinesterase as well as the effect of CPF exposure on serum PON1 in workers occupationally exposed to CPF. Saliva, blood and urine were collected from agricultural workers (n=120) from Egypt's Menoufia Governorate to determine PON1 genotype, blood cholinesterase activity, serum PON1 activity towards chlorpyrifos-oxon (CPOase) and paraoxon (POase), and urinary levels of the CPF metabolite 3,5,6-trichloro-2-pyridinol (TCPy). The PON1 55 (P≤0.05) but not the PON1 192 genotype had a significant effect on CPOase activity. However, both the PON1 55 (P≤0.05) and PON1 192 (P≤0.001) genotypes had a significant effect on POase activity. Workers had significantly inhibited AChE and BuChE after CPF application; however, neither CPOase activity nor POase activity was associated with ChE depression when adjusted for CPF exposure (as determined by urinary TCPy levels) and stratified by PON1 genotype. CPOase and POase activity were also generally unaffected by CPF exposure although there were alterations in activity within specific genotype groups. Together, these results suggest that workers retained the capacity to detoxify chlorpyrifos-oxon under the exposure conditions experienced by this study population regardless of PON1 genotype and activity and that effects of CPF exposure on PON1 activity are minimal

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA

CD39 activity correlates with stage and inhibits platelet reactivity in chronic lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>Chronic lymphocytic leukemia (CLL) is characterized by accumulation of mature appearing lymphocytes and is rarely complicated by thrombosis. One possible explanation for the paucity of thrombotic events in these patients may be the presence of the ecto-nucleotidase CD39/NTDPase-1 on the surface of the malignant cells in CLL. CD39 is the major promoter of platelet inhibition <it>in vivo </it>via its metabolism of ADP to AMP. We hypothesize that if CD39 is observed on CLL cells, then patients with CLL may be relatively protected against platelet aggregation and recruitment and that CD39 may have other effects on CLL, including modulation of the disease, via its metabolism of ATP.</p> <p>Methods</p> <p>Normal and malignant lymphocytes were isolated from whole blood from patients with CLL and healthy volunteers. Enzyme activity was measured via radio-TLC assay and expression via FACS. Semi-quantititative RT-PCR for CD39 splice variants and platelet function tests were performed on several samples.</p> <p>Results</p> <p>Functional assays demonstrated that ADPase and ATPase activities were much higher in CLL cells than in total lymphocytes from the normal population on a per cell basis (p-value < 0.00001). CD39 activity was elevated in stage 0–2 CLL compared to stage 3–4 (p < 0.01). FACS of lymphocytes demonstrated CD39 expression on > 90% of normal and malignant B-lymphocytes and ~8% of normal T-lymphocytes. RT-PCR showed increased full length CD39 and splice variant 1.5, but decreased variant 1.3 in CLL cells. Platelet function tests showed inhibition of platelet activation and recruitment to ADP by CLL cells.</p> <p>Conclusion</p> <p>CD39 is expressed and active on CLL cells. Enzyme activity is higher in earlier stages of CLL and decreased enzyme activity may be associated with worsening disease. These results suggest that CD39 may play a role in the pathogenesis of malignancy and protect CLL patients from thrombotic events.</p

Fibroblast and Epidermal Cell-Type I Collagen Interactions: Cell Culture and Human Studies

Fibroblast and epidermal cell-type I collagen sponge interactions were studied in cell culture as well as in humans. In cell culture, fibroblasts were observed to migrate and proliferate throughout a type I collagen sponge containing either hyaluronic acid (HA) or fibronectin (FN). Fibroblasts accumulated in the center of the pores in sponges containing HA and appeared to surround themselves with newly synthesized extracellular matrix. In sponges containing FN, fibroblasts attached to and elongated along the collagen fibers of the sponge. In the absence of FN or HA protein synthesis of fibroblasts appeared to be inhibited by the presence of the type I collagen sponge. Epidermal cells grown on plastic or on type I collagen, formed sheets. Epidermal cells grown on a collagen sponge morphologically appeared different than cells grown on plastic. The type I collagen matrix studied in cell culture was applied to dermal wounds of patients with pressure ulcers in order to evaluate its effect on dermal wound healing. The areas of ulcers treated for 6 weeks with a type I collagen sponge decreased by about 40% compared with no change in the areas of untreated controls. Preliminary results suggest that a type I collagen sponge is a biocornpatible substrate with fibroblasts and epidermal cells and may be effective in enhancing healing of chronic skin ulcers

Empirical entropic contributions in computational docking: Evaluation in APS reductase complexes

The results from reiterated docking experiments may be used to evaluate an empirical vibrational entropy of binding in ligand–protein complexes. We have tested several methods for evaluating the vibrational contribution to binding of 22 nucleotide analogues to the enzyme APS reductase. These include two cluster size methods that measure the probability of finding a particular conformation, a method that estimates the extent of the local energetic well by looking at the scatter of conformations within clustered results, and an RMSD-based method that uses the overall scatter and clustering of all conformations. We have also directly characterized the local energy landscape by randomly sampling around docked conformations. The simple cluster size method shows the best performance, improving the identification of correct conformations in multiple docking experiments. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2008Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60220/1/20936_ftp.pd

Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning

Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation

A consistent derivation of the quark--antiquark and three quark potentials in a Wilson loop context

In this paper we give a new derivation of the quark-antiquark potential in the Wilson loop context. This makes more explicit the approximations involved and enables an immediate extension to the three-quark case. In the $q\overline{q}$ case we find the same semirelativistic potential obtained in preceding papers but for a question of ordering. In the $3q$ case we find a spin dependent potential identical to that already derived in the literature from the ad hoc and non correct assumption of scalar confinement. Furthermore we obtain the correct form of the spin independent potential up to the $1/m^2$ order.Comment: 30 pages, Revtex (3 figures available as hard copies only), IFUM 452/F