Translation termination depends on the sequential ribosomal entry of eRF1 and eRF3.

Translation termination requires eRF1 and eRF3 for polypeptide-and tRNA-release on stop codons. Additionally, Dbp5/DDX19 and Rli1/ABCE1 are required; however, their function in this process is currently unknown. Using a combination of in vivo and in vitro experiments, we show that they regulate a stepwise assembly of the termination complex. Rli1 and eRF3-GDP associate with the ribosome first. Subsequently, Dbp5-ATP delivers eRF1 to the stop codon and in this way prevents a premature access of eRF3. Dbp5 dissociates upon placing eRF1 through ATP-hydrolysis. This in turn enables eRF1 to contact eRF3, as the binding of Dbp5 and eRF3 to eRF1 is mutually exclusive. Defects in the Dbp5-guided eRF1 delivery lead to premature contact and premature dissociation of eRF1 and eRF3 from the ribosome and to subsequent stop codon readthrough. Thus, the stepwise Dbp5-controlled termination complex assembly is essential for regular translation termination events. Our data furthermore suggest a possible role of Dbp5/DDX19 in alternative translation termination events, such as during stress response or in developmental processes, which classifies the helicase as a potential drug target for nonsense suppression therapy to treat cancer and neurodegenerative diseases

Conformation-dependent GAD65 autoantibodies in diabetes

Aims/hypothesis. Conformation-dependent autoantibodies directed against GAD65 are markers of Type 1 diabetes. In this study we aimed to determine whether the substitution of GAD65 with GAD67 amino acids would affect the binding of conformation-dependent GAD65 autoantibodies. Methods. We used PCR-based site-directed mutagenesis to generate a series of mutated GAD65 cDNA constructs in which specific GAD65 coding sequences for regions of the protein critical for autoantibody binding were replaced with GAD67 coding sequences. Results. The introduction of a point mutation at position 517, substituting glutamic acid with proline, markedly reduced the binding of disease-associated GAD65 antibodies. The binding of GAD65 antibodies to the E517P mutant was reduced in the sera of all newly diagnosed Type 1 diabetes patients (n=85) by a mean of 72% (p<0.0001) compared with binding to wild-type GAD65. Patients with latent autoimmune diabetes in adults (n=24) showed a similar reduction in binding (79% reduction, p<0.0001). First-degree relatives who subsequently progressed to Type 1 diabetes (n=12) showed a reduction in binding of 80% compared with a reduction of only 65% among relatives who had not progressed to disease (n=38; p=0.025). In healthy GAD65Ab-positive individuals who did not progress to diabetes during a 9-year follow-up period (n=51), binding to GAD65-E517P was reduced by only 28% compared with binding to wild-type GAD65. Conclusions/interpretation. Differences in autoantibody binding to wild-type GAD65 versus GAD65-E517P may provide predictive information about Type 1 diabetes risk beyond that provided by the presence or absence of GAD65 autoantibodies. Lack of binding to mutant GAD65-E517P defines GAD65-positive individuals who are at higher risk of developing diabetes

Association Study of TRPC4 as a Candidate Gene for Generalized Epilepsy with Photosensitivity

Photoparoxysmal response (PPR) is characterized by abnormal visual sensitivity of the brain to photic stimulation. Frequently associated with idiopathic generalized epilepsies (IGEs), it might be an endophenotype for cortical excitability. Transient receptor potential cation (TRPC) channels are involved in the generation of epileptiform discharges, and TRPC4 constitutes the main TRPC channel in the central nervous system. The present study investigated an association of PPR with sequence variations of the TRPC4 gene. Thirty-five single nucleotide polymorphisms (SNP) within TRPC4 were genotyped in 273 PPR probands and 599 population controls. Association analyses were performed for the broad PPR endophenotype (PPR types I-IV; n = 273), a narrow model of affectedness (PPR types III and IV; n = 214) and PPR associated with IGE (PPR/IGE; n = 106) for each SNP and for corresponding haplotypes. Association was found between the intron 5 SNP rs10507456 and PPR/IGE both for single markers (P = 0.005) and haplotype level (P = 0.01). Three additional SNPs (rs1535775, rs10161932 and rs7338118) within the same haplotype block were associated with PPR/IGE at P < 0.05 (uncorrected) as well as two more markers (rs10507457, rs7329459) located in intron 3. Again, the corresponding haplotype also showed association with PPR/IGE. Results were not significant following correction for multiple comparisons by permutation analysis for single markers and Bonferroni-Holm for haplotypes. No association was found between variants in TRPC4 and other phenotypes. Our results showed a trend toward association of TRPC4 variants and PPR/IGE. Further studies including larger samples of photosensitive probands are required to clarify the relevance of TRPC4 for PPR and IGE

Relationships of cochlear coiling shape and hearing frequencies in cetaceans, and the occurrence of infrasonic hearing in Miocene Mysticeti

Baleen whales Mysticeti are known to use low frequencies LF; 200 Hz and below and infrasound lt; 20 Hz for communication. The lowest hearing limits of toothed whales Odontoceti , which are able to produce ultrasound gt; 20 kHz , reach low frequencies. Researchers have tried to understand the evolution of LF and infrasonic hearing in mysticetes by linking the shape of the inner ear cochlea or individual cochlear measurements to known hearing frequencies and making inferences to extinct species. Using landmark based shape analysis of complete cochlear coiling, we show that cochlear coiling shape correlates with LF and high frequency HF; gt; 10 kHz hearing limits in cetaceans. Very LF 50 Hz and infrasonic hearing are associated with, for example, a protruding second turn, a descending apex, and a high number of turns. Correlations between cochlear and cranial variables and cochlear and cranial shape indicate that low LF hearing limits are furthermore connected to longer cochleae and relatively larger cranial widths. Very LF hearing in Mysticeti appeared in the middle Miocene, and mysticete infrasonic hearing had evolved by the late Miocene. Complete cochlear coiling is suitable for estimating hearing limits in cetaceans, closely approximated by cochlear length times number of cochlear turn

Detection of autoantibodies against reactive oxygen species modified glutamic acid decarboxylase-65 in type 1 diabetes associated complications

<p>Abstract</p> <p>Background</p> <p>Autoantibodies against glutamate decarboxylase-65 (GAD₆₅Abs) are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD₆₅Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD₆₅Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD₆₅in these three different GAD₆₅Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD₆₅(ROS-GAD₆₅) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD₆₅(ROS-GAD₆₅Abs) and quantitative assays in T1D associated complications.</p> <p>Results</p> <p>From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD₆₅as compared to native GAD₆₅(N-GAD₆₅). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD₆₅. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10^-6M) followed by nephropathic (1.81 × 10^-6M) and uncomplicated (3.11 × 10^-7M) T1D patients for ROS-GAD₆₅compared to N-GAD₆₅.</p> <p>Conclusion</p> <p>Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD₆₅that induce increased production of ROS-GAD₆₅Abs. Hence regulation of ROS-GAD₆₅Abs could offer novel tools for analysing and possibly treating T1D complications.</p

Sequence Analysis of the Cloned mRNA Coding for Glyceraldehyde-3-Phosphate Dehydrogenase from Chicken Heart Muscle

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66279/1/j.1432-1033.1983.tb07239.x.pd

Long-Lived Plasma Cells and Memory B Cells Produce Pathogenic Anti-GAD65 Autoantibodies in Stiff Person Syndrome

Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases