Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Kmfor ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Kmfor ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches

Comparison of ankle-brachial pressure index and pulse wave velocity as markers of cognitive function in a community-dwelling population

<p>Abstract</p> <p>Background</p> <p>Vascular factors have been implicated in the development of cognitive decline and dementia. The purpose of this study is to determine the association of the Ankle Brachial pressure Index (ABI) and brachial-ankle Pulse Wave Velocity (ba-PWV) to cognitive impairment in a community-dwelling population.</p> <p>Methods</p> <p>The ABI and ba-PWV were measured using the volume-plethymographic apparatus in 388 subjects aged 60 years old and over. The Mini-Mental State Examination was also employed to measure global cognitive status. The effectiveness of the ABI and ba-PWV as putative markers of cognitive impairment were determined by using a multiple logistic regression analysis after adjusting for confounding factors.</p> <p>Results</p> <p>Subjects with poor cognition were significantly older and less well educated than those with normal cognition. According to the multiple logistic regression analysis, the lowest ABI tertile was found to be a significant independent risk factor (OR = 3.19, 95% CI = 1.30 to 7.82) of the cognitive impairment, whereas the highest brachial-ankle PWV tertile was not.</p> <p>Conclusions</p> <p>A low ABI was an independent risk factor for cognitive impairment in community-dwelling older populations, whereas a high ba-PWV may not be. Further research will be required to analyze ABI and PWV with greater accuracy.</p

Common Sole Larvae Survive High Levels of Pile-Driving Sound in Controlled Exposure Experiments

In view of the rapid extension of offshore wind farms, there is an urgent need to improve our knowledge on possible adverse effects of underwater sound generated by pile-driving. Mortality and injuries have been observed in fish exposed to loud impulse sounds, but knowledge on the sound levels at which (sub-)lethal effects occur is limited for juvenile and adult fish, and virtually non-existent for fish eggs and larvae. A device was developed in which fish larvae can be exposed to underwater sound. It consists of a rigid-walled cylindrical chamber driven by an electro-dynamical sound projector. Samples of up to 100 larvae can be exposed simultaneously to a homogeneously distributed sound pressure and particle velocity field. Recorded pile-driving sounds could be reproduced accurately in the frequency range between 50 and 1000 Hz, at zero to peak pressure levels up to 210 dB re 1µPa2 (zero to peak pressures up to 32 kPa) and single pulse sound exposure levels up to 186 dB re 1µPa2s. The device was used to examine lethal effects of sound exposure in common sole (Solea solea) larvae. Different developmental stages were exposed to various levels and durations of pile-driving sound. The highest cumulative sound exposure level applied was 206 dB re 1µPa2s, which corresponds to 100 strikes at a distance of 100 m from a typical North Sea pile-driving site. The results showed no statistically significant differences in mortality between exposure and control groups at sound exposure levels which were well above the US interim criteria for non-auditory tissue damage in fish. Although our findings cannot be extrapolated to fish larvae in general, as interspecific differences in vulnerability to sound exposure may occur, they do indicate that previous assumptions and criteria may need to be revised

Genome-wide association trans-ethnic meta-analyses identifies novel associations regulating coagulation Factor VIII and von Willebrand Factor plasma levels

BACKGROUND: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF. METHODS: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events. RESULTS: We identified 13 novel genome-wide significant ( P≤2.5×10-8) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk. CONCLUSIONS: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events

Evidence for Genetic Overlap Between Schizophrenia and Age at First Birth in Women

IMPORTANCE: A recently published study of national data by McGrath et al in 2014 showed increased risk of schizophrenia (SCZ) in offspring associated with both early and delayed parental age, consistent with a U-shaped relationship. However, it remains unclear if the risk to the child is due to psychosocial factors associated with parental age or if those at higher risk for SCZ tend to have children at an earlier or later age. OBJECTIVE: To determine if there is a genetic association between SCZ and age at first birth (AFB) using genetically informative but independently ascertained data sets. DESIGN, SETTING, AND PARTICIPANTS: This investigation used multiple independent genome-wide association study data sets. The SCZ sample comprised 18 957 SCZ cases and 22 673 controls in a genome-wide association study from the second phase of the Psychiatric Genomics Consortium, and the AFB sample comprised 12 247 genotyped women measured for AFB from the following 4 community cohorts: Estonia (Estonian Genome Center Biobank, University of Tartu), the Netherlands (LifeLines Cohort Study), Sweden (Swedish Twin Registry), and the United Kingdom (TwinsUK). Schizophrenia genetic risk for each woman in the AFB community sample was estimated using genetic effects inferred from the SCZ genome-wide association study. MAIN OUTCOMES AND MEASURES: We tested if SCZ genetic risk was a significant predictor of response variables based on published polynomial functions that described the relationship between maternal age and SCZ risk in offspring in Denmark. We substituted AFB for maternal age in these functions, one of which was corrected for the age of the father, and found that the fit was superior for the model without adjustment for the father's age. RESULTS: We observed a U-shaped relationship between SCZ risk and AFB in the community cohorts, consistent with the previously reported relationship between SCZ risk in offspring and maternal age when not adjusted for the age of the father. We confirmed that SCZ risk profile scores significantly predicted the response variables (coefficient of determination R2 = 1.1E-03, P = 4.1E-04), reflecting the published relationship between maternal age and SCZ risk in offspring by McGrath et al in 2014. CONCLUSIONS AND RELEVANCE: This study provides evidence for a significant overlap between genetic factors associated with risk of SCZ and genetic factors associated with AFB. It has been reported that SCZ risk associated with increased maternal age is explained by the age of the father and that de novo mutations that occur more frequently in the germline of older men are the underlying causal mechanism. This explanation may need to be revised if, as suggested herein and if replicated in future studies, there is also increased genetic risk of SCZ in older mothers

Proton Magnetic Resonance Spectroscopy in 22q11 Deletion Syndrome

OBJECTIVE: People with velo-cardio-facial syndrome or 22q11 deletion syndrome (22q11DS) have behavioral, cognitive and psychiatric problems. Approximately 30% of affected individuals develop schizophrenia-like psychosis. Glutamate dysfunction is thought to play a crucial role in schizophrenia. However, it is unknown if and how the glutamate system is altered in 22q11DS. People with 22q11DS are vulnerable for haploinsufficiency of PRODH, a gene that codes for an enzyme converting proline into glutamate. Therefore, it can be hypothesized that glutamatergic abnormalities may be present in 22q11DS. METHOD: We employed proton magnetic resonance spectroscopy ((1)H-MRS) to quantify glutamate and other neurometabolites in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of 22 adults with 22q11DS (22q11DS SCZ+) and without (22q11DS SCZ-) schizophrenia and 23 age-matched healthy controls. Also, plasma proline levels were determined in the 22q11DS group. RESULTS: We found significantly increased concentrations of glutamate and myo-inositol in the hippocampal region of 22q11DS SCZ+ compared to 22q11DS SCZ-. There were no significant differences in levels of plasma proline between 22q11DS SCZ+ and 22q11DS SCZ-. There was no relationship between plasma proline and cerebral glutamate in 22q11DS. CONCLUSION: This is the first in vivo(1)H-MRS study in 22q11DS. Our results suggest vulnerability of the hippocampus in the psychopathology of 22q11DS SCZ+. Altered hippocampal glutamate and myo-inositol metabolism may partially explain the psychotic symptoms and cognitive impairments seen in this group of patients

The role of reactive oxygen species in apoptosis of the diabetic kidney

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney

Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion

The Shareholder Wealth Effects of Insurance Securitization: Preliminary Evidence from the Catastrophe Bond Market

Insurance securitization has long been hailed as an important tool to increase the underwriting capacity for companies exposed to catastrophe-related risks. However, global volumes of insurance securitization have remained surprisingly low to date which raises questions over its benefits. In this paper, we examine changes in the market value of insurance and reinsurance firms which announce their engagement in insurance securitization by issuing catastrophe (Cat) bonds. Consistent with the hitherto underwhelming contribution of Cat bonds to global catastrophe coverage, we do not find evidence that Cat bonds lead to strong wealth gains for shareholders in the issuing firm. More importantly, we report large variations in the distribution of wealth effects in response to the issue announcement. We show that the wealth effects for shareholders in firms which issue Cat bonds appear to be driven by explanations according to which Cat bonds offer cost savings relative to other forms of catastrophe risk management (and less by the potential of Cat bonds to hedge catastrophe risk). Thus, abnormal returns are particularly large for issues by firms which face low levels of loss uncertainty (which reduces the information acquisition costs in financial markets) as well as for issues during periods when prices for catastrophe coverage (including Cat bonds) are low