307 research outputs found
Identification and Characterization of Novel Mutations in the Human Gene Encoding the Catalytic Subunit Calpha of Protein Kinase A (PKA)
The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser109 significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly186 completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly186 is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg2+, which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser109 or Gly186 may be faced with abnormal development and possibly severe disease
Measurement of the Electric Form Factor of the Neutron at Q^2 = 0.3-0.8 (GeV/c)^2
The electric form factor of the neutron, G_En, has been measured at the Mainz
Microtron by recoil polarimetry in the quasielastic D(e_pol,e'n_pol)p reaction.
Three data points have been extracted at squared four-momentum transfers Q^2 =
0.3, 0.6 and 0.8 (GeV/c)^2. Corrections for nuclear binding effects have been
applied.Comment: 9 pages, 7 figures, 2 tables. Accepted for publication in EPJ
Deletion of the GABAA α2-subunit does not alter self dministration of cocaine or reinstatement of cocaine seeking
Rationale
GABAA receptors containing α2-subunits are highly represented in brain areas that are involved in motivation and reward, and have been associated with addiction to several drugs, including cocaine. We have shown previously that a deletion of the α2-subunit results in an absence of sensitisation to cocaine.
Objective
We investigated the reinforcing properties of cocaine in GABAA α2-subunit knockout (KO) mice using an intravenous self-administration procedure.
Methods
α2-subunit wildtype (WT), heterozygous (HT) and KO mice were trained to lever press for a 30 % condensed milk solution. After implantation with a jugular catheter, mice were trained to lever press for cocaine (0.5 mg/kg/infusion) during ten daily sessions. Responding was extinguished and the mice tested for cue- and cocaine-primed reinstatement. Separate groups of mice were trained to respond for decreasing doses of cocaine (0.25, 0.125, 0.06 and 0.03 mg/kg).
Results
No differences were found in acquisition of lever pressing for milk. All genotypes acquired self-administration of cocaine and did not differ in rates of self-administration, dose dependency or reinstatement. However, whilst WT and HT mice showed a dose-dependent increase in lever pressing during the cue presentation, KO mice did not.
Conclusions
Despite a reported absence of sensitisation, motivation to obtain cocaine remains unchanged in KO and HT mice. Reinstatement of cocaine seeking by cocaine and cocaine-paired cues is also unaffected. We postulate that whilst not directly involved in reward perception, the α2-subunit may be involved in modulating the “energising” aspect of cocaine’s effects on reward-seeking
A Measurement of the Electric Form Factor of the Neutron through at (GeV/c)
We report the first measurement of the neutron electric form factor
via using a solid polarized target. was
determined from the beam-target asymmetry in the scattering of longitudinally
polarized electrons from polarized deuterated ammonia, ND. The
measurement was performed in Hall C at Thomas Jefferson National Accelerator
Facility (TJNAF) in quasi free kinematics with the target polarization
perpendicular to the momentum transfer. The electrons were detected in a
magnetic spectrometer in coincidence with neutrons in a large solid angle
segmented detector. We find at (GeV/c).Comment: Latex2e 5 pages, 3 figure
Allosteric Inhibition of Parkinson's-Linked LRRK2 by Constrained Peptides
Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson's Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.</p
Positional Cloning of Zinc Finger Domain Transcription Factor Zfp69, a Candidate Gene for Obesity-Associated Diabetes Contributed by Mouse Locus Nidd/SJL
Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT–PCR, and RACE–PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lepob background, the diabetogenic Zfp69SJL allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes
Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2
Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson’s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD
Nucleon Charge and Magnetization Densities from Sachs Form Factors
Relativistic prescriptions relating Sachs form factors to nucleon charge and
magnetization densities are used to fit recent data for both the proton and the
neutron. The analysis uses expansions in complete radial bases to minimize
model dependence and to estimate the uncertainties in radial densities due to
limitation of the range of momentum transfer. We find that the charge
distribution for the proton is significantly broad than its magnetization
density and that the magnetization density is slightly broader for the neutron
than the proton. The neutron charge form factor is consistent with the Galster
parametrization over the available range of Q^2, but relativistic inversion
produces a softer radial density. Discrete ambiguities in the inversion method
are analyzed in detail. The method of Mitra and Kumari ensures compatibility
with pQCD and is most useful for extrapolating form factors to large Q^2.Comment: To appear in Phys. Rev. C. Two new figures and accompanying text have
been added and several discussions have been clarified with no significant
changes to the conclusions. Now contains 47 pages including 21 figures and 2
table
The deuteron: structure and form factors
A brief review of the history of the discovery of the deuteron in provided.
The current status of both experiment and theory for the elastic electron
scattering is then presented.Comment: 80 pages, 33 figures, submited to Advances in Nuclear Physic
Correction to: Febrile illness in high-risk children: a prospective, international observational study
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