The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.
Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a lipid anchor. Six members of this family have been identified in mammals (GPC1-GPC6). Glypicans act as regulators of the activity of various cytokines, including Wnts, Hedgehogs, and bone morphogenetic proteins. It has been reported that processing by a convertase is required for GPC3 activity during convergent extension in zebrafish embryos, for GPC3-induced regulation of Wnt signaling, and for the binding of GPC3 to Wnt5a. In our laboratory, we have recently demonstrated that GPC3 promotes the growth of hepatocellular carcinomas (HCCs) by stimulating canonical Wnt signaling. Because there is increasing evidence indicating that the structural requirements for GPC3 activity are cell type specific, we decided to investigate whether GPC3 needs to be processed by convertases to stimulate cell proliferation and Wnt signaling in HCC cells. We report here that a mutant GPC3 that cannot be processed by convertases is still able to play its stimulatory role in Wnt activity and HCC growth.Glypicans are a family of heparan sulfate proteoglycans that are linked to the exocytoplasmic surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor (1-4). To date six glypicans have been identified in mammals (GPC1-GPC6) (4, 5). In general, glypicans are expressed predominantly during development. Their expression levels change in a stage-and tissue-specific manner, suggesting that they are involved in morphogenesis (4, 6).Genetic and functional studies performed in Drosophila, Xenopus, zebrafish, and mammals have demonstrated that glypicans can stimulate the signaling activity of Wnts, Hedgehogs, and bone morphogenetic proteins (7-17). In the specific case of Wnts, glypicans have been reported to stimulate both the canonical and non-canonical pathways (8,13,14,18). Because Wnts are known to bind to heparan sulfate (19), it was originally proposed that the stimulatory activity of glypicans is based on their ability to act as facilitators of the interaction between Wnts and their receptors (8). This hypothesis has been supported by the finding that several Wnts can co-immunoprecipitate with glypicans (14,15,18,20,21), although it is now known that at least in certain cell systems the heparan sulfate chains are not required for the glypicanWnt interaction (18,21). This lack of requirement of the heparan sulfate chains for the interaction with a growth factor has also been reported for perlecan, another heparan sulfate proteoglycan that can regulate cell proliferation (22). In addition to acting as facilitators of ligand-receptor interactions, glypicans have been shown to play a role during development in the transport of Wnts, Hedgehogs, and bone morphogenetic proteins for the purpose of morphogen gradient formation (23-28).One of the highly conserved structural features of the glypicans is the location of the insertion sites for the heparan sulfate chains, which seem to be restricted to the last 55 amino acids, placing such ...
Background:Living donor kidney transplantation (LDKT) has several advantages over deceased donor kidney transplantation. Yet rates of living donation are declining in Canada and there exists significant interprovincial variability. Efforts to improve living donation tend to focus on the patient and barriers identified at their level, such as not knowing how to ask for a kidney or lack of education. These efforts favor those who have the means and the support to find living donors. Thus, a Canadian Institutes of Health Research (CIHR)-organized workshop recommended that education efforts to understand and remove barriers should focus on health professionals (HPs). Despite this, little attention has been paid to what they identify as barriers to discussing LDKT with their patients.Objective:Our aim was to explore HP-identified barriers to discuss living donation with patients in 3 provinces of Canada with low (Quebec), moderate (Ontario), and high (British Columbia) rates of LDKT.Design:This study consists of an interpretive descriptive approach as it enables to move beyond description and inform clinical practice.Setting:Purposive criterion and quota sampling were used to recruit HPs from Quebec, Ontario, and British Columbia who are involved in the care of patients with kidney disease and/or with transplant coordination.Patients:Not applicable.Measurements:Semistructured interviews were conducted. The interview guide was developed based on a preliminary analytical framework and a review of the literature.Methods:Thematic analysis was used to analyze the data stemming from the interviews. The coding process comprised of a deductive and inductive approach, and the use of a qualitative analysis software (NVivo 11). Following this, themes were identified and developed. Interviews were conducted until thematic saturation was obtained. In total, we conducted 16 telephone interviews as thematic saturation was attained.Results:Six predominant themes emerged: (1) lack of communication between transplant and dialysis teams, (2) absence of referral guidelines, (3) role perception and lack of multidisciplinary involvement, (4) HP’s lack of information and training, (5) negative attitudes of some HP toward LDKT, (6) patient-level barriers as defined by the HP. HPs did mention patients’ attitudes and some characteristics as the main barriers to discussions about living donation; this was noted in all provinces. HPs from Ontario and British Columbia indicated multiple strategies being implemented to address some of these barriers. Those from Ontario mentioned strategies that center on the core principles of provincial-level standardization, while those from British Columbia center on engaging the entire multidisciplinary team and improved role perception. We noted a dearth of such efforts in Quebec; however, efforts around education and promotion, while tentative, have emerged.Limitations:Social desirability and selection bias. Our analysis might not be applicable to other provinces.Conclusions:HPs involved with the referral and...
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