Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles

The destabilistion of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline→leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca<SUP>2+</SUP>] and result in death of cells

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties

Can psychological models bridge the gap between clinical guidelines and clinicians' behaviour?:A randomised controlled trial of an intervention to influence dentists' intention to implement evidence-based practice

Objective The lag between publication of evidence for clinical practice and implementation by clinicians may be decades. Research using psychological models demonstrates that changing intention is very important in changing behaviour. This study examined an intervention (rehearsing alternative actions) to change dentists' intention to implement evidence-based practice (EBP) for third molar (TM) management.Design Randomised controlled trial/postal.Setting Primary care.Subjects and methods Dentists were randomly selected from the Scottish Dental Practice Board Register, then randomly allocated to intervention or control groups, and sent a questionnaire. The intervention group listed management alternatives to TM extraction prior to their TM extraction intention, and the control group did not. Based on psychological models for reducing a behaviour's frequency (EBP is weighted against TM extraction), prior listing of alternatives should decrease extraction intention.Main outcome measure Intention to extract TMs.Results A total of 99 dentists - 70 Males, 29 Females; mean age = 41.42 years (SD = 8.62) participated in the study. The intervention significantly influenced intention to extract TMs, as desired. Despite similar background and knowledge of management alternatives, participants in the intervention group had significantly lower intention to extract: control group mean (SD) = 0.39 (1.99); intervention group mean (SD) = - 0.78 (1.89); mean difference (SE) = 1.17 (0.42); 95% confidence interval for the difference = 0.34 to 1.99.Conclusion Results suggest this intervention, which successfully influenced a proximal predictor of behaviour pertinent to dental EBP, may result in improved EBP in a service-level trial. Basing implementation interventions and trial methodology on psychological models may effectively bridge the gap between clinical guidelines and practice.</p