Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Dilated Cardiomyopathy Decreases Protein Stability,Increases Protein Unfolding,and Perturbs Protein Structure,but Does Not Affect Protein Function

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein''s overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.     

Children's Social and Cognitive Development and Child-Care Quality: Testing for Differential Associations Related to Poverty,Gender, or Ethnicity

This paper addresses some of the implicit rules that may be involved in scientific inquiry. Factors outside the scientific method such as personal characteristics, belief systems, and scholarly eminence may play a role in scientific inquiry. In this case study, we show that the referencing of two prominent psychologists, Jean Piaget and Clark Hull, declined sharply after they died. This change, we suggest, may be due to the absence of an actual influence on colleagues and students.     

Glutamatergic Postsynaptic Density Protein Dysfunctions in Synaptic Plasticity and Dendritic Spines Morphology: Relevance to Schizophrenia and Other Behavioral Disorders Pathophysiology,and Implications for Novel Therapeutic Approaches

Emerging researches point to a relevant role of postsynaptic density (PSD) proteins, such as PSD-95, Homer, Shank, and DISC-1, in the pathophysiology of schizophrenia and autism spectrum disorders. The PSD is a thickness, detectable at electronic microscopy, localized at the postsynaptic membrane of glutamatergic synapses, and made by scaffolding proteins, receptors, and effector proteins; it is considered a structural and functional crossroad where multiple neurotransmitter systems converge, including the dopaminergic, serotonergic, and glutamatergic ones, which are all implicated in the pathophysiology of psychosis. Decreased PSD-95 protein levels have been reported in postmortem brains of schizophrenia patients. Variants of Homer1, a key PSD protein for glutamate signaling, have been associated with schizophrenia symptoms severity and therapeutic response. Mutations in Shank gene have been recognized in autism spectrum disorder patients, as well as reported to be associated to behaviors reminiscent of schizophrenia symptoms when expressed in genetically engineered mice. Here, we provide a critical appraisal of PSD proteins role in the pathophysiology of schizophrenia and autism spectrum disorders. Then, we discuss how antipsychotics may affect PSD proteins in brain regions relevant to psychosis pathophysiology, possibly by controlling synaptic plasticity and dendritic spine rearrangements through the modulation of glutamate-related targets. We finally provide a framework that may explain how PSD proteins might be useful candidates to develop new therapeutic approaches for schizophrenia and related disorders in which there is a need for new biological treatments, especially against some symptom domains, such as negative symptoms, that are poorly affected by current antipsychotics.     

H9724, a Monoclonal Antibody to Borrelia burgdorferi's Flagellin, Binds to Heat Shock Protein 60 (HSP60) Within Live Neuroblastoma Cells: A Potential Role for HSP60 in Peptide Hormone Signaling and in an Autoimmune Pathogenesis of the Neuropathy of Lyme Disease

Although Borrelia burgdorferi, the causative agent of Lyme disease, is found at the site of many disease manifestations, local infection may not explain all its features. B. burgdorferi's flagellin cross-reacts with a component of human peripheral nerve axon, previously identified as heat shock protein 60 (HSP60). The cross-reacting epitopes are bound by a monoclonal antibody to B. burgdorferi's flagellin, H9724. Addition of H9724 to neuroblastoma cell cultures blocks in vitro spontaneous and peptide growth-factor–stimulated neuritogenesis. Withdrawal of H9724 allows return to normal growth and differentiation. Using electron microscopy, immunoprecipitation and immunoblotting, and FACS analysis we sought to identify the site of binding of H9724, with the starting hypotheses that the binding was intracellular and not identical to the binding site of II-13, a monoclonal anti-HSP60 antibody. The current studies show that H9724 binds to an intracellular target in cultured cells with negligible, if any, surface binding. We previously showed that sera from patients with neurological manifestations of Lyme disease bound to human axons in a pattern identical to H9724's binding; these same sera also bind to an intracellular neuroblastoma cell target. II-13 binds to a different HSP60 epitope than H9724; II-13 does not modify cellular function in vitro. As predicted, II-13 bound to mitochondria, in a pattern of cellular binding very different from H9724, which bound in a scattered cytoplasmic, nonorganelle-related pattern. H9724's effect is the first evidence that HSP60 may play a role in peptide-hormone–receptor function and demonstrates the modulatory potential of a monoclonal antibody on living cells.