Allergenicity assessment of genetically modified crops—what makes sense?

GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops

Autism diagnosis differentiates neurophysiological responses to faces in adults with tuberous sclerosis complex

- Background: Autism spectrum disorder (ASD) is a common and highly heritable neurodevelopmental disorder that is likely to be the outcome of complex aetiological mechanisms. One strategy to provide insight is to study ASD within tuberous sclerosis complex (TSC), a rare disorder with a high incidence of ASD, but for which the genetic cause is determined. Individuals with ASD consistently demonstrate face processing impairments, but these have not been examined in adults with TSC using event-related potentials (ERPs) that are able to capture distinct temporal stages of processing. - Methods: For adults with TSC (n = 14), 6 of which had a diagnosis of ASD, and control adults (n = 13) passively viewed upright and inverted human faces with direct or averted gaze, with concurrent EEG recording. Amplitude and latency of the P1 and N170 ERPs were measured. - Results: Individuals with TSC + ASD exhibited longer N170 latencies to faces compared to typical adults. Typical adults and adults with TSC-only exhibited longer N170 latency to inverted versus upright faces, whereas individuals with TSC + ASD did not show latency differences according to face orientation. In addition, individuals with TSC + ASD showed increased N170 latency to averted compared to direct gaze, which was not demonstrated in typical adults. A reduced lateralization was shown for the TSC + ASD groups on P1 and N170 amplitude. - Conclusions: The findings suggest that individuals with TSC + ASD may have similar electrophysiological abnormalities to idiopathic ASD and are suggestive of developmental delay. Identifying brain-based markers of ASD that are similar in TSC and idiopathic cases is likely to help elucidate the risk pathways to ASD

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Eukaryotic peptide chain initiation: A study using fluorescent probes

The minimum factors required for efficient formation of both the ternary complex (eIF-2$\cdot$GTP$\cdot$Met-tRNA\sb{\rm f}\sp{\rm Met}) and the 40S initiation complex (40S$\cdot$message$\cdot$Met-tRNA\sb{\rm f}\sp{\rm Met}) have been highly purified and characterized by filtration assays. In our proposed model, eIF-2B first catalyzes GTP/GDP exchange on eIF-2 in physiological Mg\sp{2+} concentrations (1-2 mM). Next, eIF-3 promotes ternary complex formation as well as the formation of 40S complex. Co-eIF-2C, having both eIF-2B and eIF-3 activities, can substitute the latter two factors. Co-eIF-2A also promotes ternary complex formation and inhibits mRNA-induced ternary complex dissociation. Formation of the 40S complex is enhanced when a translatable message is present (either a synthetic AUG-containing polyribonucleotide or globin mRNA). In order to further study protein-protein and protein-RNA interactions, fluorescent derivatives of GDP and ATP were developed for use in steady-state fluorescence spectroscopy experiments. The probes were synthesized by reaction of a periodate oxidized ribonucleotide and a fluorescent amine-containing derivative with subsequent hydride reduction. The structure regarding the modification to the ribose ring has been proposed based UV-vis, NMR and FAB-MS studies. Tryptophan and fluorescein labeled GDP were used as probes to observe conformational changes in eIF-2 in response to Mg\sp{2+} concentration. Fluorescein labeled ATP (ATP*F) has been shown to bind specifically and reversibly to eIF-2 with high affinity. The binding of ATP*F to eIF-2 was independent of GTP binding and was used to measure the binding of p\sp{67} (a key regulatory factor) to eIF-2 (K\sb{\rm d}\approx 1 $\mu$M). ATP*F was also used as a probe to characterize binding of eIF-2 to all the other factors involved in 40S complex formation. Using a combination of both spectroscopic and filtration assays, the binary association constants between quaternary complex (Q), message (M) and 40S ribosomes (R) were measured. The message enhancement observed in 40S complex formation was thermodynamically quantitated and characterized as a cooperative system. A 79-fold difference was calculated for K\sb{\rm a(Q,R)} and K\sb{\rm a(Q,R\cdot M)}

New Insights into an Old Problem: Ternary Complex (Met-tRNAf·eIF-2 ·GTP) Formation in Animal Cells

An early step in the initiation of protein synthesis in eukaryotic cells is the formation of a ternary complex between initiation Factor 2, Met-tRNA f and GTP; Met-tRNA f ·elF- 2 ·GTP. This complex is an obligate intermediate in the binding of Met-tRNA f to 40S ribosomes and thus is important for translation of all mRNAs. Several reports have indicated that formation and/or stability of the ternary complex in vitro may require a number of additional protein factors (for a review, see Gupta et al, 1987). For example, it has been reported that the bulk of reticulocyte eIF-2 is purified as eIF-2 ·GDP and a protein factor eIF-2B is required to displace GDP from eIF-2 ·GDP and subsequently to form ternary complex in the presence of Mg2+. Recently, Roy et al (1988) reported that two additional protein factor preparations, Co-eIF-2A and Co-eIF-2C are required for the formation of a stable ternary complex in the presence of natural mRNAs. Co-eIF-2A and Co-eIF-2C are two highly purified protein preparations that possess eIF-2 stimulatory activities described in a number of papers from Gupta and co-workers (see Gupta et al, 1987, Roy et al, 1988). Co-eIF-2A possesses a mass of 94 kDa and appears to be unrelated to any of the other known initiation factors. On the other hand, Co-eIF-2C resembles the previously described initiation factor eIF-3. Because of the considerable controversy surrounding the roles of these auxiliary factors in ternary complex formation, our three laboratory groups collaborated to compare the structures of the relevant initiation factors prepared independently and to re-examine their roles in ternary complex formation

Cloning and Characterization of Complementary DNA Encoding the Eukaryotic Initiation Factor 2-Associated 67-kDa Protein (p67)

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit

Characteristics of the Eukaryotic Initiation Factor 2 Associated 67-kDa Polypeptide

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p67) protects the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation, and this promotes protein synthesis in the presence of active eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328]. This report presents the results of studies related to characteristics of p67 action and the mechanism of p67:eIF-2 interaction: (1) p67 antibodies inhibited protein synthesis in hemin-supplemented rabbit reticulocyte lysates, and such inhibition was reversed by preincubation of the antibodies, specifically with p67. (2) p67 inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 alpha-subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 beta-subunit. (3) p67 bound specifically to the eIF-2 gamma-subunit. p67 co-immunoprecipitated with the eIF-2 subunits when a p67/eIF-2 mixture was treated with p67 or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubated specifically with the eIF-2 gamma-subunit antibodies, subsequent co-immunoprecipitation of p67 with the eIF-2 subunits was completely inhibited. Similarly, preincubation of p67 and p67 antibodies prevented subsequent p67 binding to eIF-2. Preincubation of eIF-2, with either eIF-2 alpha- or beta-subunit antibodies, had no effect on p67 co-immunoprecipitation with the eIF-2 subunits. (4) p67:eIF-2 interaction is necessary for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation