Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

Immune thrombocytopenic purpura in three patients with preexisting ulcerative colitis

Ulcerative colitis (UC) is associated with extraintestinal diseases in numerous target tissues. Associated immune-mediated hematological diseases, however, are rarely described. We report three Caucasian adult patients with UC and immune thrombocytopenic purpura (ITP). Platelet-associated antibodies (IgG) were positive in two patients, and bone marrow examinations in two patients revealed normal to increased megakaryocyte numbers. ITP was treated with corticosteroids in all patients. Two patients eventually received intravenous immune gamma-globulin, and one patient required surgical splenectomy. Of particular interest, UC preceded the onset of ITP in all patients (by from 1 to 19 yr). This suggests that ITP in these patients is causally associated with UC, possibly secondary to immunostimulation from lumenal antigens and altered immunoregulation.     

Improved method for studying skin lipid samples from cyanoacrylate strips by high-performance thin-layer chromatography

A non-invasive method for sampling skin surface lipids is cyanoacrylate stripping (CAC-TS). It was the purpose of our study to improve the method for sampling of skin surface lipids and the separation of epidermal lipid fractions by a modification of the methods described by Melnik and by Imokawa et al. Briefly, lipids on the glass slide sampled by CAC-TS from the forearm of 75 volunteers and from the forehead of 60 volunteers were eluted in hexane/ethanol under ultrasonication. Identification of the diluted total superficial sebaceous and epidermal skin lipids was performed by sequential high-performance thin-layer chromatography. For quantification of the lipids we used densitometric methods. By this modified method we were able to show a clear and complete separation of all relevant lipids from a cyanoacrylate strip that represented 1-2 mg stratum corneum only.     

Experimental evaluation of theoretical contact forces in the cat patellofemoral joint

The purpose of this study was to evaluate the accuracy of patellofemoral contact forces predicted from a planar model of the patella by comparison with experimentally determined in situ contact forces. Patellofemoral contact pressures and areas were measured experimentally in an animal preparation with pressure sensitive film. Patellar tendon forces and lines of action used as input to the model were measured in the intact joint of the same preparation. Predicted and measured contact forces at different joint loads were compared at three different joint angles using linear regression analysis. r2-coefficients ranged from 0.94 to 0.95, and the slopes of the regression lines ranged from 1.64 to 2.11 for the three joint angles. The high r2-coefficients for all comparisons indicate that both methods were able to quantify the relative changes in the cat patellofemoral contact forces under different loading conditions accurately. However, the consistent finding of slopes greater than 1.0 indicates that the measured contact forces were systematically larger than the corresponding predicted forces. Analysis of the possible sources for the observed discrepancies between predicted and measured contact forces suggested that the directly measured patellar tendon forces were the most likely candidate causing the systematic differences. The results of this study suggest that a relatively simple model of the patellofemoral joint appears to be valid to quantify joint contact forces if appropriate patellar tendon force values can be provided as input to the model.     

Speciation of arsenic oxides using laser desorption/ionization time-of-flight mass spectrometry

Positive and negative ion mass spectra of arsenic trioxide (As2O3) and arsenic pentaoxide (As2O5) have been obtained by single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 nm was used for the simultaneous desorption and ionization of the solid sample. High-mass cluster ions that are unique to the oxidation state of each oxide sample appear in the negative ion mass spectra. The As2O3 produces As3O5-, while the As2O5 yields As3O8-. The formation of unique negative cluster ions presents the capability for arsenic oxidation state speciation by laser desorption/ionization mass spectrometry. The ability of time-of-flight mass spectrometry to examine the relative amounts of each arsenic oxide present in a series of mixtures is discussed. Application of our speciation technique to a model incinerator sample is demonstrated.     

Older adult controlled drinkers and abstainers

Little is known about the lifetime course of alcohol problems, especially during late adulthood. Many individuals with a history of alcohol problems achieve remission of their symptoms through abstinence or controlled drinking. This study examined 135 older adults with a prior history of alcohol diagnoses who were symptom free for at least the past year. Two groups were identified based on their alcohol consumption within the past year: abstinent individuals (n = 92) and controlled drinkers (n = 43). The groups did not differ in age, racial composition, education, income, or years since their last alcohol-related symptom, but they did differ in gender composition, indices of alcoholism severity, history of formal and informal treatment, as well as lifetime alcohol consumption patterns. Abstinent individuals had more severe alcohol problems, consumed higher amounts of alcohol on drinking days, had more years of heavy alcohol consumption, and were more likely to have attended alcohol treatment and Alcoholics Anonymous (AA). The controlled drinkers had a longer history of moderate social drinking, and their current consumption habits appeared to be similar to symptom-free older adult drinkers. The results suggest that gender, alcoholism severity, history of formal and informal treatment, and past consumption patterns are associated with whether older adults with histories of alcoholism attain successful outcomes through abstinence or controlled drinking.     

A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation

Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-DeltaTF-PR-DeltaPol, which contains the protease linked to short native flanking sequences (DeltaTF and DeltaPol) fused to the maltose binding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina, D. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP-DeltaTF-PR-DeltaPol protein leads to the formation of the PR-DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands. We now report the purification and characterization of the PR-DeltaPol intermediate and the kinetics of its processing to the mature protease. Unlike the MBP-DeltaTF-PR-DeltaPol precursor, PR-DeltaPol has proteolytic activity similar to that of the mature enzyme at pH 5.0. The pH rate profile for kcat/Km is similar to that of the mature protease above pH 4.0. Although the PR-DeltaPol is more sensitive than the mature protease toward denaturing reagents, both the enzymatic activity and the intrinsic fluorescence of PR-DeltaPol are linearly dependent on the protein concentration, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteolytic reaction at the N terminus of the protease, the proteolytic reaction at the C terminus of the protease is second order in protein concentration. These results are discussed in terms of a mechanism in which the C-terminally located DeltaPol peptide chains are cleaved intermolecularly to release the mature protease.     

Calcium modulation of adrenocorticotropin levels in women--a clinical research center study

In vitro calcium modulation of anterior pituitary hormone secretion has been well described. In addition, several investigations performed in human subjects have documented modulation of the circulating levels of pituitary hormones by supraphysiological calcium concentrations. Recent data from our laboratory document the existence of an extracellular calcium-sensing receptor that is thought to mediate the effects of variations in extracellular calcium on the secretion of PTH and calcitonin. We have also demonstrated the presence of this receptor in pituitary-derived, ACTH-secreting AtT-20 cells as well as in the anterior pituitary of rats and mice. In the present study we investigated the effect on anterior pituitary hormone levels of variations in serum calcium within the physiological range. We serially measured serum levels of ionized calcium (Cai), ACTH, cortisol, TSH, and PRL during 90-min iv infusions (on separate days) of calcium, citrate, and dextrose in 10 healthy women with a mean age of 55 +/- 5 yr. During the calcium infusion, the serum Cai level increased significantly from 4.32 +/- 0.10 mg/dL at baseline to 4.86 +/- 0.08 mg/dL at completion (P = 0.002), and this change was accompanied by a significant increment in the serum ACTH level from 9.87 +/- 1.32 to 16.31 +/- 2.84 pg/mL (P = 0.0008). There was no change in the serum ACTH level during the citrate infusion despite significant decrements in serum Cai, nor were there changes in either Cai or ACTH during the dextrose infusion. Finally, changes in Cai did not alter TSH or PRL levels. In summary, our dynamic studies are the first to demonstrate an increase in baseline serum ACTH levels in response to physiological increments in Cai (i.e. increments within the normal range). This effect was specific for increments and not decrements in serum Cai and was selective for ACTH, as TSH and PRL levels did not change with any of the infusions.     

Naming in dementia secondary to Parkinson's, Huntington's, and Alzheimer's diseases

Confrontation naming problems have been found in patients with dementia secondary to Alzheimer's (AD), Huntington's (HD), and in a subset of Parkinson's disease (PD) patients with dementia. The source of the naming deficit has not been established. The "Perception" and the "Semantic Feature" theories have been proposed to explain this naming dysfunction. Subjects with dementia secondary to AD, HD, and PD were given three tasks to determine which theory best explained the source of confrontation naming problems. The three tasks including picture matching, visual recognition, and confrontation naming were given to 42 subjects with dementia secondary to AD, HD, and PD controlled for severity of dementia, and to age-matched controls. Subjects with dementia did not have significantly more difficulty matching pictures but did have more difficulty associating pictures through semantic features. Subjects with mild dementia secondary to AD and HD had significantly more confrontation naming errors than subjects with mild dementia secondary to PD and normal controls. All subjects with moderate dementia had significantly more confrontation naming errors than normal controls. Statistical power may have been limited due to the small number of subjects in each group. The source of the reduction in confrontation naming performance in subjects with dementia secondary to AD, HD, and PD originated in the deterioration of semantic fields. The perception theory was rejected as findings were consistent with the semantic feature theory.