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Biomedical subjects

M Altmann

Publications and source records attributed to M Altmann.

At least 37 records · Page 2Linked to original sources
PubMed1997

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.

Amino Acid Sequence↗
PubMed1997

Translation initiation factor-dependent extracts from yeast Saccharomyces cerevisiae.

Translation initiation factor-dependent extracts are prepared from Saccharomyces cerevisiae strains that have no or reduced activity of a translation initiation factor. Elimination of factor activity can be achieved by deletion of the gene encoding the factor if it is not essential for the survival of the strain. If the gene is essential it is placed under the control of the regulatable GAL1 promoter and its expression is shut off in vivo. Alternatively, a temperature-sensitive mutation can be introduced into the gene and the activity of the gene product eliminated in vitro by preincubation of the extract at the nonpermissive temperature. Factor-dependent extracts can be complemented in vitro with purified initiation factor preparations isolated from S. cerevisiae or from Escherichia coli cells expressing them from plasmid-encoded constructs. To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags. Initiation factor-dependent extracts can be used to study initiation factor structure-function relationships and initiation factor requirements for specific mRNA translation.

Gene Deletion↗
PubMed1996

Stoichiometry and site-specific phosphorylation of human progesterone receptor in native target cells and in the baculovirus expression system.

Human progesterone receptor (PR) in T47D breast cancer cells is phosphorylated on nine different serine residues; three are hormone-inducible (Ser102, Ser294, and Ser345), while others are basal but hormone-stimulated. In the present study, we have compared the phosphorylation state of native and recombinant PR expressed in a baculovirus insect cell system. Stoichiometric measurements showed that unliganded native PR in T47D cells was approximately 50% phosphorylated ( approximately 4 phosphates/PR) and became essentially 100% phosphorylated ( approximately 9 phosphates/PR) when bound to hormone. Unliganded PR expressed in Sf9 insect cells was phosphorylated with a similar stoichiometry ( approximately 3 phosphates/PR), but the phosphate content did not change with hormone addition. Site-specific phosphorylation analyzed by tryptic phosphopeptide mapping and manual peptide sequencing revealed that expressed PR bound to hormone in the Sf9 insect cells was phosphorylated on all the same sites as hormone-treated PR in T47D cells. Only minor differences were detected in the relative proportion of three sites (two basal sites and Ser345) and phosphorylation did not occur on alternate sites. Interestingly, unliganded baculovirus-expressed PR was constitutively phosphorylated on hormone inducible sites and was phosphorylated on basal sites to the same extent as hormone treated PR. Thus, in the absence of hormone, the phosphorylation state of baculovirus-expressed PR resembled that of the hyperphosphorylated native PR. In contrast to native PR, the expressed receptor in cytosols of Sf9 cells did not form a large oligomeric complex suggesting that hyperphosphorylation may be due to dissociation of the complex in the absence of hormone. This study demonstrating phosphorylation on correct sites with a stoichiometry similar to that of native PR indicates that overexpressed PR in the baculovirus system is suitable for in vitro structure/function studies.

Amino Acid Sequence↗
PubMed1996

Displacement correction and surface reconstruction of the retina using scanning laser ophthalmoscopic images.

A method for a three-dimensional surface reconstruction of the retina in the area of the papilla is presented. The surface reconstruction is based on a sequence of discrete gray-level images of the retina recorded by a scanning laser ophthalmoscope (SLO). The underlying assumption of the surface reconstruction algorithm developed here is that the depth information is also encoded in the brightness values of the single pixels in addition to the ordinary spatial 2D information. The brightness of an image position depends on the degree of reflection of a confocal laser beam. Only those surface structures located directly in the focus plane of the confocal laser beam produce a high response to the laser light. The displacements between the single images of a sequence are considered to be approximately linear and are corrected by applying the cepstrum technique. The depth is estimated from the volumetric representation of the image sequence by searching for the maximal value of the brightness within a computed depth profile, at every image position. In the resulting images, disturbances occurring during the recording cause incorrect local estimations of the depth. These local disturbances are corrected by applying specially developed surface improvement processes. The work is concluded with a comparison of several different approaches to reduce the noise and disturbances in SLO image data.

Algorithms↗
PubMed1996

Decreasing disability in the 21st century: the future effects of controlling six fatal and nonfatal conditions.

OBJECTIVES: This study assessed the effects of reducing fatal and nonfatal health conditions on the number of functionally limited older Americans in the coming decades. METHODS: Data from the 1990 census and the Longitudinal Study of Aging were used to project the number of functionally limited older Americans from 2001 to 2049, assuming 1% biennial reductions in five conditions that shorten life expectancy (coronary artery disease, stroke, cancer, diabetes, and confusion) and one condition that decreases functional ability (arthritis). RESULTS: Decreasing the prevalence of arthritis by 1% every 2 years would lead to a much greater reduction in functional limitation between 2001 and 2049 (4 million person-years) than would decreasing any of the other conditions by the same amount. Decreases in two fatal conditions (cancer and coronary artery disease) would lead to increases in functional limitation (0.9 and 0.1 million person-years, respectively). CONCLUSIONS: Advances against common nonfatal disabling conditions would be more effective than advances against fatal conditions in blunting the large increase in the functionally limited older population anticipated in the 21st century.

Aged↗
PubMed1995

The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity.

The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF-4B. We have added six histidine residues to the C-terminus of Tif3 protein (Tif3-His6p) and purified the tagged protein by affinity chromatography. Tif3-His6p stimulates translation and mRNA binding to ribosomes in a Tif3-dependent in vitro system. Furthermore, it binds to single-stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro. In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF-4B. A role for Tif3/eIF-4B and RNA annealing activity in the scanning process is proposed.

Animals↗
PubMed1995

Isolation of a protein complex containing translation initiation factor Prt1 from Saccharomyces cerevisiae.

Translation initiation factor Prt1 was purified from a ribosomal salt wash fraction of Saccharomyces cerevisiae cells by ammonium sulfate precipitation, DEAE chromatography, phosphocellulose chromatography, sucrose density gradient centrifugation, and non-denaturing polyacrylamide gel electrophoresis. Prt1 protein cofractionates with four other polypeptides during all steps of purification suggesting that it is part of a protein complex containing polypeptide subunits with apparent molecular masses of 130, 80, 75 (Prt1), 40, and 32 kDa. Deletion of the first AUG codon in the published sequence of the PRT1 gene results in the synthesis of functional Prt1 protein indicating that the actual molecular mass of the Prt1 subunit is 82.7 kDa. This is in agreement with results from primer extension experiments reported earlier by Keierleber et al. (Keierleber, C., Wittekind, M., Qin, S., and McLaughlin, C. S. (1986) Mol. Cell. Biol. 6, 4419-4424). The Prt1-containing protein complex is an active translation factor as shown by its ability to restore translation in a cell-free system derived from a temperature-sensitive prt1 mutant strain in which endogenous Prt1 activity is inactivated by heating the extract to 37 degrees C. The question of whether the Prt1-containing protein complex represents the yeast homologue of mammalian translation initiation factor eIF-3 is discussed.

Base Sequence↗
PubMed1995

Susceptible-infected-removed epidemic models with dynamic partnerships.

The author extends the classical, stochastic, Susceptible-Infected-Removed (SIR) epidemic model to allow for disease transmission through a dynamic network of partnerships. A new method of analysis allows for a fairly complete understanding of the dynamics of the system for small and large time. The key insight is to analyze the model by tracking the configurations of all possible dyads, rather than individuals. For large populations, the initial dynamics are approximated by a branching process whose threshold for growth determines the epidemic threshold, R0, and whose growth rate, lambda, determines the rate at which the number of cases increases. The fraction of the population that is ever infected, omega, is shown to bear the same relationship to R0 as in models without partnerships. Explicit formulas for these three fundamental quantities are obtained for the simplest version of the model, in which the population is treated as homogeneous, and all transitions are Markov. The formulas allow a modeler to determine the error introduced by the usual assumption of instantaneous contacts for any particular set of biological and sociological parameters. The model and the formulas are then generalized to allow for non-Markov partnership dynamics, non-uniform contact rates within partnerships, and variable infectivity. The model and the method of analysis could also be further generalized to allow for demographic effects, recurrent susceptibility and heterogeneous populations, using the same strategies that have been developed for models without partnerships.

Communicable Diseases↗
PubMed1995

Simulation of stochastic micropopulation models--IV. SNAPPERS: model implementation for genetic traits.

The current paper concerning stochastic micropopulation simulations describes SNAPPERS, which serves as a framework for simulation models of the genetic transmission of disease. The versions described are implemented using the simulation shell, SUMMERS, which includes the generic commonalities of several micropopulation models. Population members in SNAPPERS move through states related to the individual's status relative to the genotype (phenotype). Features of the model include one or two major loci, polygenic and common familial contribution to the phenotype, assortative mating, and flexibility in defining gene action. The user can select from multiple ascertainment strategies for analysis of simulated families.

Chromosome Mapping↗
PubMed1995

Progesterone receptor and the mechanism of action of progesterone antagonists.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Interaction of the progesterone receptor with binding proteins for FK506 and cyclosporin A.

T47D human breast carcinoma cells and the chicken oviduct were used to study the structure of the nonactivated progesterone receptor (PR) complex. Immunoprecipitation of PR (B form) from cytosol extracts was performed using monoclonal antibody PR6, a cross-reactive antibody prepared to chicken PR. Analysis of the PR complex by sodium dodecyl sulfate gels and Western immuno-blotting revealed the presence of several specific copurifying proteins. Consistent with previous reports, the two heat shock proteins, hsp90 and hsp70, were shown to be present. A third 59-kilodalton (kDa) protein observed previously was confirmed to be p59 (also called hsp56 or FKBP52), which has been shown to bind the immunosuppressant drug FK506. Two additional PR-associated proteins were observed that had not been previously recognized with human PR. These have molecular masses of 54-kDa and 23-kDa and have been shown by Western blotting to be related to the proteins p54 and p23 that are associated with chicken PR. P23 is a novel protein of unknown function and p54 or FKBP54 has been recently shown to be another FK506-binding protein related to p59. Finally, the cyclosporin A-binding protein, CyP-40, could be detected in isolated chicken PR complexes and in PR complexes that were reconstituted in vitro, but this protein was not detected in human PR complexes, which are less stable than chicken PR complexes in cytosol extracts. The functional significance of FK506 and cyclosporin A-binding proteins to hormone action was tested using a T47D cell line that contained a progestin reporter gene, MMTV-CAT. Treatment with cyclosporin A had no effect on the basal level of CAT expression, but it caused a dramatic increase in the sensitivity and magnitude of the response to the synthetic progestin, R5020. The enhanced response elicited by drug treatment was blocked by the antiprogestin RU486 indicating that this effect was receptor-mediated. While cyclosporin A enhanced progestin action in T47D cells, it inhibited a PR/reporter gene system in L cells. The drugs FK506 and rapamycin had no effect on progestin action in T47D cells, but they stimulated glucocorticoid action in T47D cells. Thus, the effects of these immunosuppressant drugs vary with the cell type and hormonal system that is tested. Whether these drug effects relate directly to the immunophilins bound in receptor complexes remains unknown.

Animals↗
PubMed1994

Network analytic methods for epidemiological risk assessment.

The authors measure the efficacy of three methods for predicting the time to infection for susceptible individuals in a population undergoing an HIV epidemic. The methods differ in whether they require detailed information of the contact network and whether they require knowledge of the initial source of infection. Efficacy is evaluated using simulations for 20 different contact patterns. Only the risk score that uses both kinds of information accounts for more than 15 per cent of individual variability. The efficacy of this score ranges from 10 per cent in very unstructured populations to 60 per cent for spatially localized contact networks. This improved performance may be explained by the larger fraction of the total variability not due to the disease dynamics. When all variables are dichotomized, the two poorer methods produce odds ratios between 1.4 and 2.3. The odds ratio for the risk score with full information ranges from 2.5 to 17. Risk assessment protocols and intervention programmes are encouraged to assess contact patterns and detect sources of infection.

Analysis of Variance↗
PubMed1994

A clarification of the phi mixing model.

The authors review a deterministic model proposed for the analysis of two-way contingency tables that arise in counts of pairwise interactions. This model decomposes the table into the sum of two matrices with special forms: in one the contacts are distributed selectively, in the other they are distributed at random. We show that this model has several inherent problems. The decomposition is not unique, which compromises estimation and interpretation of the parameters; the deterministic framework provides no basis for estimation or hypothesis testing; and the assumption of decomposibility is supported by neither empirical evidence nor theoretical considerations. We show that generalized linear models provide a suitable alternative once the probability process is specified and the overparameterization is removed.

Animals↗
PubMed1994

The yeast Saccharomyces cerevisiae system: a powerful tool to study the mechanism of protein synthesis initiation in eukaryotes.

This review summarizes recent progress in the study of initiation of protein synthesis in the yeast Saccharomyces cerevisiae. Biochemical and genetic approaches provide new insight into the recognition of the 5'-end of mRNA by initiation factors and 40S ribosomes, unwinding of mRNA secondary structures in the untranslated region and proper recognition of the AUG start codon. Experiments with initiation factor-dependent cell-free systems have facilitated studies of factor functions and factor requirements for translation of different mRNAs. The analysis of mutations which suppress the inhibitory effect on translation of RNA secondary structure in the 5'-untranslated region of yeast mRNAs has led to the identification of gene products which may be involved in both transcription and translation.

Eukaryotic Cells↗
PubMed1993

A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity.

The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract. In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.

Amino Acid Sequence↗
PubMed1993

Simulation of stochastic micropopulation models--I. The SUMMERS simulation shell.

A generic, abstract model and the simulation shell based on it, both called SUMMERS, are used as a framework for the implementation of stochastic micropopulation models; in these, each individual is followed separately while moving through a sequence of states. The shell supports groups of interacting members, individual characteristics and multiple simultaneous activities. Stochastic decisions may be made using Monte Carlo rules. Keywords control the simulations and the reports generated. A sensitivity analysis utility allows assessment of the dependency of outcomes on model features. Extensive use has been made of software engineering techniques. Specializations of SUMMERS are described in subsequent papers.

Data Collection↗