Translation initiation and surface plasmon resonance: new technology applied to old questions.

Translation initiation in eukaryotes is a complicated process involving some of the largest cellular structures, the ribosomes, together with approx. 11 initiation factors, and a poorly characterized set of other proteins. The concerted action of all these components ultimately results in the formation of an 80 S ribosomal complex on the AUG codon of an mRNA, which is competent to start polypeptide production. In this brief overview, we describe the strategies developed by our laboratory to apply surface plasmon resonance (SPR)-based technology to the problem of elucidating kinetic aspects of substeps within the translation-initiation reaction. We then review how other groups have used similar SPR-based techniques to study related interactions.

A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties.

The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA. We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E. We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1. eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell. The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures. Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10(2)-times weaker than eIF4E1 in vitro. The eIF4E2 cap-binding affinity is in the typical micromolar range. The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions. Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.

The effects of routine strength on adaptation and information search in recurrent decision making.

The strength of decision routines was manipulated within a computer-controlled micro world simulation involving recurrent decision making. During a learning phase, participants were led to prefer a certain brand of an industrial good either about 15 times in a weak routine or about 30 times in a strong routine condition. In the test phase of Experiment 1, participants were confronted with changes in the microworld that rendered the routine obsolete. Routine maintenance over a series of repeated acquisition decisions was assessed as the major dependent variable. Although new information clearly suggested that a deviation from the routine would be beneficial, strong routine participants were more likely to maintain the routine compared to weak routine participants and a control group in which a comparable option (same outcome probabilities as the routine) carried an unfamiliar brand label. Experiment 2 investigated the effects of routine strength on information search. After having learned the routine, participants were asked to make one final decision involving the routine. The task was either framed as being similar to the learning task or as being novel. Before making the final decision, participants were asked to consider new information about the alternatives. Strong routine participants in the familiar task condition preferred information that favored the routine and avoided unfavorable information. If the task was framed as being novel, such confirmation biases disappeared completely. In contrast, weak routine participants exhibited a moderate confirmation bias in their searches independent from task framing.

Temporal updating scheme for probabilistic neural network with application to satellite cloud classification--further results.

A novel temporal updating approach for probabilistic neural network classifiers was developed by Tian et al. (2000) to account for temporal changes of spectral and temperature features of clouds in the visible and infrared GOES 8 (Geostationary Operational Environmental Satellite) imagery data. In this paper, a new method referred to as moving singular value decomposition (MSVD) is introduced to improve the classification rate of the boundary blocks or blocks containing cloud types with non-uniform texture. The MSVD method is then incorporated into the temporal updating scheme and its effectiveness is demonstrated on several sequences of GOES 8 cloud imagery data. These results indicate that the incorporation of the new MSVD improves the overall performance of the temporal updating process by almost 10%

Stabilization of eukaryotic initiation factor 4E binding to the mRNA 5'-Cap by domains of eIF4G.

The eukaryotic cap-binding complex eIF4F is an essential component of the translational machinery. Recognition of the mRNA cap structure through its subunit eIF4E is a requirement for the recruitment of other translation initiation factors to the mRNA 5'-end and thereby for the attachment of the 40 S ribosomal subunit. In this study, we have investigated the mechanistic basis of the observation that eIF4E binding to the cap is enhanced in the presence of the large eIF4F subunit, eIF4G. We show that eIF4E requires access to both the mRNA 5'-cap and eIF4G to form stable complexes with short RNAs. This stabilization can be achieved using fragments of eIF4G that contain the eIF4E binding site but not the RNA recognition motifs. Full-length eIF4G is shown to induce increased eIF4E binding to cap analogues that do not contain an RNA body. Both results show that interaction of eIF4G with the mRNA is not necessary to enhance cap binding by eIF4E. Moreover, we show that the effect of binding of full-length eIF4G on the cap affinity of eIF4E can be further modulated through binding of Pab1 to eIF4G. These data are consistent with a model in which heterotropic cooperativity underlies eIF4F function.

Temporal updating scheme for probabilistic neural network with application to satellite cloud classification.

In cloud classification from satellite imagery, temporal change in the images is one of the main factors that causes degradation in the classifier performance. In this paper, a novel temporal updating approach is developed for probabilistic neural network (PNN) classifiers that can be used to track temporal changes in a sequence of images. This is done by utilizing the temporal contextual information and adjusting the PNN to adapt to such changes. Whenever a new set of images arrives, an initial classification is first performed using the PNN updated up to the last frame while at the same time, a prediction using Markov chain models is also made based on the classification results of the previous frame. The results of both the old PNN and the predictor are then compared. Depending on the outcome, either a supervised or an unsupervised updating scheme is used to update the PNN classifier. Maximum likelihood (ML) criterion is adopted in both the training and updating schemes. The proposed scheme is examined on both a simulated data set and the Geostationary Operational Environmental Satellite (GOES) 8 satellite cloud imagery data. These results indicate the improvements in the classification accuracy when the proposed scheme is used.

Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.

Translational repression by human 4E-BP1 in yeast specifically requires human eIF4E as target.

4E-binding proteins (4E-BPs) are believed to have important regulatory functions in controlling the rate of translation initiation in mammalian cells. They do so by binding to the mRNA cap-binding protein, eIF4E, thereby inhibiting formation of the cap-binding complex, a process essential for cap-dependent translation initiation. We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find its activity to be dependent on substitution of human eIF4E for its yeast counterpart. Translation initiation and growth are inhibited when human 4E-BP1 is expressed in a strain with the human eIF4E substitution, but not in an unmodified strain. We have compared the relative affinities of human 4E-BP1 for human and yeast eIF4E, both in vitro using an m7GTP cap-binding assay and in vivo using a yeast two-hybrid assay, and find that the affinity of human 4E-BP1 for human eIF4E is markedly greater than for yeast eIF4E. Thus yeast eIF4E lacks structural features required for binding to human 4E-BP1. These results therefore demonstrate that the features of eIF4E required for binding to 4E-BP1 are distinct from those required for cap-complex assembly.

A study of cloud classification with neural networks using spectral and textural features.

The problem of cloud data classification from satellite imagery using neural networks is considered in this paper. Several image transformations such as singular value decomposition (SVD) and wavelet packet (WP) were used to extract the salient spectral and textural features attributed to satellite cloud data in both visible and infrared (IR) channels. In addition, the well-known gray-level cooccurrence matrix (GLCM) method and spectral features were examined for the sake of comparison. Two different neural-network paradigms namely probability neural network (PNN) and unsupervised Kohonen self-organized feature map (SOM) were examined and their performance were also benchmarked on the geostationary operational environmental satellite (GOES) 8 data. Additionally, a postprocessing scheme was developed which utilizes the contextual information in the satellite images to improve the final classification accuracy. Overall, the performance of the PNN when used in conjunction with these feature extraction and postprocessing schemes showed the potential of this neural-network-based cloud classification system.

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

Toward an understanding of the fluorescence intensity changes observed on fluorescein 5'-Isothiocyanate-Na(+),K (+)-ATPase.

The fluorescence emission intensity between the Na(+), and the K(+) complex of Na(+),K(+)-ATPase, labeled with fluorescein 5'-isothiocyanate (FITC), differs by 30 to 40%. Experimental studies are carried out to elucidate the physical reasons which account this intensity difference. The dissociation constant of protolysis of the covalently bound FITC and its fluorescence decay times are determined in media of different ionic compositions and are compared with the corresponding properties of a synthetic model compound. The fluorophore bound to the protein is characterized by two decay times in the nanosecond range; the model compound, by a single one. The static fluorescence intensity changes are discussed on the basis of these results.

Initial solar irradiance determinations from nimbus 7 cavity radiometer measurements.

Preliminary results from solar radiation measurements from the earth radiation budget experiment on the Nimbus 7 satellite yield a mean value of 1376.0 watts per square meter for the "solar constant" from 16 November 1978 to 15 May 1979. The observed variability (root-mean-square deviation) is +/- 0.73 watt per square meter (+/- 0.05 percent) for the period.

Satellite Observations of the Earth's Radiation Budget.

Meteorological satellites have provided the first complete data on energy exchange between earth and space. The planetary albedo is 29 percent for the mean annual case, and the entire earth-atmosphere system is in near radiative equilibrium. More energy is absorbed in tropical regions than previously believed, and major energy source and sink regions exist within latitude belts.