ABSTRACT
OBJECTIVES: To determine the incidence of hypovitaminosis D in proximal femur fracture (PFF) patients and investigate whether sociodemographic factors or radiographic parameters are associated with vitamin D levels. METHODS: This is a consecutive case series of South-East Queensland patients presenting with low-energy PFFs. Vitamin D levels and sociodemographic factors (age, sex, postcode, medications and type of residence) were collected from medical records. Radiographic parameters included PFF type and cortical thickness of the femur. RESULTS: A total of 313 patients were included (mean age = 79.5 years), and 105 (34%) were deficient in vitamin D (<50 nmol/L). There was no association between vitamin D levels and sociodemographic factors or radiographic parameters. Eighty-four (84%) of vitamin D-deficient patients were not taking vitamin D supplements. CONCLUSIONS: Social and demographic factors are not correlated with vitamin D levels in this cohort. Routine vitamin D supplementation may be indicated in ageing patients although it is not always protective of low-energy fractures.
Subject(s)
Hip Fractures , Vitamin D Deficiency , Aged , Femur , Humans , Queensland/epidemiology , Vitamin D , Vitamin D Deficiency/diagnosis , Vitamin D Deficiency/epidemiologyABSTRACT
Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50) for both CRP and collagen.
Subject(s)
Protein Tyrosine Phosphatase, Non-Receptor Type 6/chemistry , Receptors, Immunologic/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Biotinylation , Blood Platelets/metabolism , COS Cells , Cricetinae , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Sequence Data , Mutagenesis , Peptides/chemistry , Phospholipase C gamma/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Syk Kinase , Tyrosine/chemistryABSTRACT
Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.
Subject(s)
Blood Platelets/metabolism , NADP/analogs & derivatives , Platelet Activation/physiology , Blood Platelets/drug effects , Calcium/metabolism , Calcium Signaling/physiology , Carbolines/pharmacology , Carrier Proteins/metabolism , Humans , NADP/metabolism , Peptides/metabolism , Piperazines/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Thrombin/pharmacologyABSTRACT
It is widely recognized that gain- and loss-of-function approaches are essential for understanding the functions of specific genes, and such approaches would be particularly valuable in studies involving human embryonic stem (hES) cells. We describe a simple and efficient approach using lipofection to transfect hES cells, which enabled us to generate hES cell lines expressing naturally fluorescent green or red proteins without affecting cell pluripotency. We used these cell lines to establish a means of diminishing gene function using small interfering (si)RNAs, which were effective at knocking down gene expression in hES cells. We then demonstrated that stable expression of siRNA could knock down the expression of endogenous genes. Application of these gain- and loss-of-function approaches should have widespread use, not only in revealing the developmental roles of specific human genes, but also for their utility in modulating differentiation.
