A difference in the rate of ribosomal elongation balances the synthesis of eukaryotic translation initiation factor (eIF)-2 alpha and eIF-2 beta

Eukaryotic translation initiation factor 2 (eIF-2) is a heterotrimer composed of three subunits designated alpha, beta, and gamma. These proteins exist in equimolar amounts in the cell and have not been detected as isolated subunits. Our research examines the basis of their balanced synthesis. Northern analysis of K562 cell mRNA revealed that eIF-2 beta was five times more abundant than eIF-2 alpha. However, immunoprecipitation of pulse-labeled K562 cells showed an equimolar rate of synthesis of eIF-2 alpha and -beta despite the 5-fold difference in the size of their mRNA pools. Addition of equal amounts of synthetic capped mRNA for eIF-2 alpha and eIF-2 beta to an in vitro translation reaction produced five times more eIF-2 alpha protein than eIF-2 beta. Determination of the polysome profile for alpha and beta mRNA in K562 cells indicated eIF-2 alpha was translated more efficiently than eIF-2 beta. Substitution of either the initiation codon context or the leader of the beta mRNA for that of alpha had only a minor effect on the translational efficiency of beta. Comparison of the rate of ribosomal elongation for the two mRNAs indicated that ribosomes associated with the beta mRNA elongate at a rate 4-fold less than that of eIF-2 alpha. Thus, the balanced translation of alpha and beta mRNA is primarily the result of a 4-fold difference in the rate of ribosomal elongation.     

Characterization of an adduct and its degradation product produced by the reaction of cyanoethylene oxide with deoxythymidine and DNA

Cyanoethylene oxide (CEO), the putative toxic and carcinogenic metabolite of acrylonitrile, is a direct-acting mutagen. CEO reacted with deoxythymidine (dT) to form a single adduct (approximately 3% dT modified). Using two-dimensional NMR spectroscopy and fast atom bombardment mass spectrometry, this adduct was identified as N3-(2-cyano-2-hydroxyethyl)deoxythymidine. Subsequently, degradation of the adduct yielded N3-(2,2-dihydroxyethyl)deoxythymidine, a hydrated form of N3-(oxoethyl)deoxythymidine. N3-(2-cyano-2-hydroxyethyl)deoxythymidine was also detected in the reaction of [2,3-14C]CEO with calf thymus DNA. Small UV peaks, not present in the control, were detected from the reaction of CEO with dA, dG and dC. However, neither their retention times nor spectral characteristics corresponded with the standards used in this study. Characterization of this cyano-hydroxyethyl adduct and its degradation product following in vitro exposure of nucleosides to CEO may provide insight as to the types of adducts that could be assessed as biomarkers in vivo, and the modifications responsible for the mutational effects of CEO.     

Stability and change in the psychological adjustment of mothers of children and adolescents with cystic fibrosis and sickle cell disease

Found moderate stability in the classification of maternal adjustment in two longitudinal studies of mothers of children and adolescents with cystic fibrosis and sickle cell disease. In terms of the transactional stress and coping model, stable poor maternal adjustment was associated with higher levels of appraisal of daily stress and palliative coping and low levels of family supportiveness. With initial levels of maternal adjustment, demographic parameters, and follow-up interval controlled, concurrent levels of daily stress accounted for significant portions of variance in maternal adjustment at follow-up for both illness groups. In addition, illness severity, child psychological adjustment, and family conflict added significant increments to maternal adjustment at follow-up in the cystic fibrosis group. Findings are discussed in terms of a basis for subsequent intervention studies to enhance the adjustment of mothers of children with chronic illness.     

Effect of sputum induction on arterial oxygen saturation and spirometry in HIV-infected patients

We wanted to study the effect of sputum induction on forced expiratory volume in one second (FEV1) and on oxygen saturation in normal controls, asymptomatic human immunodeficiency virus (HIV)-seropositive individuals, and HIV-seropositive patients under investigation for suspected Pneumocystis carinii pneumonia (PCP). Over a five month period, sputum induction with ultrasonically nebulized 3% saline was performed on 110 HIV-seropositive patients with suspected PCP, 10 asymptomatic HIV-seropositive patients, and 15 normal controls. Oxygen saturation (peak, trough and change in oxygen saturation (delta O2)) was measured throughout the procedure using pulse oximetry, and these results compared with the chest radiograph and the final pulmonary diagnosis. In addition, the effect of sputum induction on FEV1 was measured in the 15 control subjects and 10 asymptomatic HIV-seropositive patients. Compared with bronchoalveolar lavage, sputum induction had a diagnostic sensitivity for PCP of 76%. Chest radiography was 79% sensitive, and had specificity of 83%. Patients with PCP had lower peak and trough oxygen saturation values compared with the non-PCP group (mean peak 95 vs 97%; mean trough 88 vs 91%), and greater falls in O2 saturation during the procedure (mean delta O2 7.6 vs 5.5%). One subject desaturated to 76%, requiring supplemental oxygen. Sputum induction caused significant but temporary falls in FEV1 both in control and HIV-seropositive groups (mean maximum fall in FEV1 10.4 vs 12.5%). We conclude that although sputum induction causes significant falls in oxygen saturation and FEV1, it remains sensitive and safe, and provides a useful alternative to bronchoscopy for the diagnosis of PCP.     

A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp 120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of ecdysteroid action in Malacosoma disstria cells: cloning selected regions of E75- and MHR3-like genes

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.     

Within-session patterns of self-injury as indicators of behavioral function

Functional analysis assessment procedures have proven to be highly useful in identifying variables maintaining aberrant behavior like self-injury. When successful, assessments can lead to more effective treatment than when behavioral function is unknown. Because of practical limitations, not all clinicians can conduct extensive and thorough analyses prior to treatment implementation. Although relatively brief assessments have proven successful in a number of published studies, it is unclear under what conditions those assessments would match the results of a more extended analysis. This study examined a method for assessing the behavioral function of severe self-injury in four adult participants. For each participant, the initial assessment involved analyzing within-session trends and fluctuations in rates of self-injury by plotting the frequency within each minute of a session. The results of these analyses were then compared to a set of more traditional, longer-term functional analyses conducted with each participant. Results suggested that within-session analyses are viable procedures for the assessment of self-injury. Potential benefits of within-session analyses over other brief assessments and longer-term analyses are discussed.