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PubMed1991

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform.

The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.

Amino Acid Sequence↗
PubMed1991

Conserved segmental expression of Krox-20 in the vertebrate hindbrain and its relationship to lineage restriction.

The zinc-finger gene Krox-20 is expressed in two alternating segments, rhombomeres (r) 3 and 5, in the developing mouse hindbrain. This expression pattern is established prior to rhombomere formation in the mouse, but it is not known how the timing of expression relates to cellular events of segmentation, such as lineage restriction. We have cloned Krox-20 sequences from Xenopus and the chick and shown that its alternating expression pattern is conserved in these systems, suggesting that its role in hindbrain development is conserved. Analysis of the early stages of Krox-20 expression in the chick show that both domains of expression precede the restriction of cell lineage to specific rhombomeres, consistent with a role of this gene in early events of hindbrain segmentation. The finding that expression is not coincident with lineage restriction indicates that early expression may not reflect an irreversible commitment of cells to r3 and r5 and/or may be mosaic.

Amino Acid Sequence↗
PubMed2005

Evaluation and treatment of coronary artery disease in patients with end-stage renal disease.

Evaluation and treatment of coronary artery disease in patients with end-stage renal disease. Patients with end-stage renal disease (ESRD) are at increased risk of death from coronary artery disease (CAD). The metabolic milieu that results from renal dysfunction appears to accelerate the atherosclerotic process by decades in patients with ESRD. The extremely high prevalence of atherosclerosis in patients with ESRD mandates risk factor identification and treatment. Traditionally, CAD in this patient population has been treated conservatively. Analysis of large databases has highlighted the scope and complexity of this problem; nonetheless, there is a paucity of randomized, controlled trials of CAD in patients with ESRD. In this paper the following issues related to evaluation and treatment of patients with chronic kidney disease are addressed: (1) optimal CAD risk management; (2) evaluation for CAD in patients with ESRD, including the identification of coronary calcification; (3) treatment of CAD with medical therapy and revascularization; (4) relative merits of percutaneous coronary intervention versus bypass surgery. In general, an aggressive approach to medical management of CAD is warranted, even in the setting of subclinical CAD. A low threshold for diagnostic testing should be employed in patients with ESRD. When significant CAD is identified, ESRD patients appear to benefit more from revascularization compared to conservative medical management. Thus, if clinically reasonable, patients with ESRD and CAD should be managed aggressively to improve survival and reduce the incidence of future cardiac events.

Adrenergic beta-Antagonists↗
PubMed1993

Molecular analysis of a ring chromosome X in a family with fragile X syndrome.

The phenotypically normal sister of a patient affected by fragile X syndrome was referred for genetic counselling and was found to carry a mosaic karyotype 46,X,r(X)/45,X. Because the probability of the simultaneous chance occurrence of fragile X syndrome and a ring chromosome X in the same family is very low, we postulated that the breakpoint of the ring chromosome X originated in the cytogenetic break in Xq27.3 responsible for fragile X syndrome. In order to determine the relative positions of the breakpoint on the ring chromosome X and the (CGG)n unstable sequence responsible for the fragile X mutation, we used molecular markers to analyse the telomeric regions of chromosome X in this family. The results showed that the ring chromosome X was the maternal fragile X chromosome and that the telomeric deletion on the long arm encompassed the (CGG)n sequence. This suggests that the cytogenetic break in Xq27.3 is distinct from the unstable (CGG)n sequence, or that the break followed by the end-to-end fusion creating the ring chromosome was not completely conservative. Analysis of DNA markers on the short arm of chromosome X evidenced a deletion of a large part of the pseudoautosomal region, allowing us to position the genes involved in stature and in some syndromes associated with telomeric deletions of Xp on the proximal side of the pseudoautosomal region.

Adult↗
PubMed2004

Do proteins always benefit from a stability increase? Relevant and residual stabilisation in a three-state protein by charge optimisation.

The vast majority of our knowledge on protein stability arises from the study of simple two-state models. However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether the energetics of native/intermediate equilibria is well represented in current knowledge. We consider here that the overall conformational stability of three-state proteins is made of a "relevant" term and a "residual" one, corresponding to the free energy differences of the native to intermediate (N-to-I) and intermediate to denatured (I-to-D) equilibria, respectively. The N-to-I free energy difference is considered to be the relevant stability because protein-unfolding intermediates are likely devoid of biological activity. We use surface charge optimisation to first increase the overall (N-to-D) stability of a model three-state protein (apoflavodoxin) and then investigate whether the stabilisation obtained is realised into relevant or into residual stability. Most of the mutations designed from electrostatic calculations or from simple sequence conservation analysis produce large increases in the overall stability of the protein. However, in most cases, this simply leads to similarly large increases of the residual stability. Two mutations, nevertheless, show a different trend and increase the relevant stability of the protein substantially. When all the mutations are mapped onto the structure of the apoflavodoxin thermal-unfolding intermediate (obtained independently by equilibrium phi-analysis and NMR) they cluster perfectly so that the mutations increasing the relevant stability appear in the small unstructured region of the intermediate and the others in the native-like region. This illustrates the need for specific investigation of N-to-I equilibria and the structure of protein intermediates, and indicates that it is possible to rationally stabilise a protein against partial unfolding once the structure of the intermediate conformation is known, even if at low resolution.

Anabaena↗
PubMed1993

Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) mediates intracellular sorting of lysosomal enzymes, binding lysosomal enzymes in the Golgi and delivering them to a lysosomal compartment. The receptor also mediates endocytosis of extracellular ligands. We have devised a new method that rigorously measures function of the CI-MPR in intracellular sorting and used it to identify a previously uncharacterized signal near the COOH terminus of the receptor that is required for sorting. We stably transfect mutant receptors into CI-MPR-deficient mouse L cells, isolate homogeneous clonal cell lines that express a range of receptor levels for each mutant, and assay each cell line for levels of receptor expression and secretion of total phosphorylated lysosomal enzymes. Examination of the secretion phenotype of the cells as a function of receptor levels provides a sensitive indicator of the intrinsic sorting efficiency of each mutant receptor. We find that chimeric CI-MPRs that contain the bovine extracytoplasmic domain and the human or mouse transmembrane and cytoplasmic domains function identically to the bovine receptor, thus demonstrating that sorting signals are conserved. Analysis of a series of truncation and alanine scanning mutants reveals that a consensus casein kinase II site followed by 2 leucines near the COOH terminus that has the sequence (-10)DDSDEDLL(-3) is important for receptor function in sorting of lysosomal enzymes.

Amino Acid Sequence↗
PubMed2002

A candidate vaccine against influenza virus intensively improved the immunogenicity of a neutralizing epitope.

BACKGROUND: The hemagglutinin (HA) of influenza viruses is one of the major targets of the humoral response. The role of serum antibody to HA in the protection against infection has been demonstrated by long-standing observation. In previous studies, we suggested that an epitope vaccine might be a new strategy against the virus. METHODS: HA sequences of 491 H3 subtype strains from the influenza sequence database were compared and analyzed. To acquire information on the immunogenicity of the F3 epitope, F3-epitope-specific antibody levels in 81 patient sera infected with influenza virus were tested by ELISA. Based on the theory of the epitope vaccine, we designed an epitope peptide F3 (C-KAYSNCYPYDVPDY-G-KAYSNCYPYDVPDY), which contains the repeated F3 epitope KAYSNCYPYDVPDY (aa92-105) on HA (H3N2). The specificity and the titer of the antibodies induced by the epitope vaccine were determined by ELISA. The neutralizing activities of these anti-F3 antibodies were shown by inhibiting influenza virus infection of MDCK cells. RESULTS AND CONCLUSION: Comparison of HA sequences of 491 H3 subtype strains indicates that this epitope is highly conservative. Analysis of the sera from influenza virus-infected patients revealed a very low level of F3 epitope-specific antibodies, suggesting the poor immunogenicity of the F3 epitope on influenza virus. The epitope vaccine based on the F3 epitope induced high levels of F3 epitope-specific antibodies recognizing the epitope peptide F3 (antibody titer in antisera up to 1:25,600). Besides, the antisera could also recognize the natural HA in Western blotting. Interestingly, these antisera induced by the epitope vaccine could inhibit infection of MDCK cells by influenza virus (strain A/Wuhan/359/95) in the neutralization assay. These results suggest that the epitope vaccine can intensively increase the immunogenicity of neutralizing epitopes and may provide a new way to develop an effective vaccine against influenza virus.

Animals↗
PubMed1998

Evolution of gene order and chromosome number in Saccharomyces, Kluyveromyces and related fungi.

The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence database. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactislmarxianus species assemblage.

Ascomycota↗
PubMed1998

T cell receptor peptide vaccination in rheumatoid arthritis: a placebo-controlled trial using a combination of Vbeta3, Vbeta14, and Vbeta17 peptides.

OBJECTIVE: Restricted T cell receptor (TCR) gene usage has been demonstrated in animal models of autoimmune disease and has resulted in the successful use of TCR peptide therapy in animal studies. This clinical trial was undertaken to determine the safety and efficacy of a combination of Vbeta3, Vbeta14, and Vbeta17 TCR peptides in Freund's incomplete adjuvant (IFA) in patients with rheumatoid arthritis (RA). METHODS: A double-blind, placebo-controlled, multicenter, phase II clinical trial was undertaken using IR501 therapeutic vaccine, which consists of a combination of 3 peptides derived from TCRs (Vbeta3, Vbeta14, and Vbeta17) in IFA. A total of 99 patients with active RA received either 90 microg (n = 31) or 300 microg (n = 35) of IR501 or IFA alone (n = 33) as a control. The study medication and placebo were administered as a single intramuscular injection (1 ml) at weeks 0, 4, 8, and 20. RESULTS: Treatment with IR501 was safe and well tolerated. None of the patients discontinued the trial because of treatment-related adverse events. Efficacy was measured according to the American College of Rheumatology 20% improvement criteria. Using these criteria, patients in both IR501 dosage groups showed improvement in disease activity. In the most conservative analysis used to evaluate efficacy, an intent-to-treat analysis including all patients who enrolled, the 90-microg dosage group showed a statistically significant improvement compared with control patients at the 20-week time point after the third injection. Trends toward improvement were shown in both the 90-microg and the 300-microg dosage groups at week 24 after the fourth injection. CONCLUSION: IR501 therapeutic vaccine therapy was safe and well tolerated, immunogenic, and demonstrated clinical improvement in RA patients. Additional clinical trials are planned to confirm and extend these observations.

Adult↗
PubMed2006

Rad9, an evolutionarily conserved gene with multiple functions for preserving genomic integrity.

The Rad9 gene is evolutionarily conserved. Analysis of the gene from yeast, mouse and human reveal roles in multiple, fundamental biological processes primarily but not exclusively important for regulating genomic integrity. The encoded mammalian proteins participate in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, and apoptosis. Other functions include a role in embryogenesis, the transactivation of multiple target genes, co-repression of androgen-induced transcription activity of the androgen receptor, a 3'-5' exonuclease activity, and the regulation of ribonucleotide synthesis. Analyses of the functions of Rad9, and in particular its role in regulating and coordinating numerous fundamental biological activities, should not only provide information about the molecular mechanisms of several individual cellular processes, but might also lend insight into the more global control and coordination of what at least superficially present as independent pathways.

Animals↗
PubMed2006

Seasonal confounding in air pollution and health time-series studies: effect on air pollution effect estimates.

A major statistical challenge in air pollution and health time-series studies is to adequately control for confounding effects of time-varying covariates. Daily health outcome counts are most commonly analysed by Poisson regression models, adjusted for overdispersion, with air pollution levels included as a linear predictor and smooth functions for calendar time and weather variables to adjust for time-varying confounders. Various smoothers have been used so far, but the optimal strategy for choosing smoothers and their degree of smoothing remains controversial. In this work, we evaluate the performance of various smoothers with different criteria for choosing the degree of smoothing in terms of bias and efficiency of the air pollution effect estimate in a simulation study. The evaluated approaches were also applied to real mortality data from 22 European cities. The simulation study imitated a multi-city study. Data were generated from a fully parametric model. Model selection methods which optimize prediction may lead to increased biases in the air pollution effect estimate. Minimization of the absolute value of the sum of the partial autocorrelation function of the model's residuals (PACF), as a criterion to choose the degree of smoothness, gave the smallest biases. The penalized splines (PS) method with a large number of effective dfs (e.g. 8-12 per year) could be used as the basic, relatively conservative, analysis whereas the PS and natural splines in combination with PACF could be applied to provide a reasonable range of the effect estimate.

Air Pollution↗
PubMed2000

Structure of the human ubiquitin fusion gene Uba80 (RPS27a) and one of its pseudogenes.

Ubiquitin is a highly conserved 76 amino acid protein that is generated in the cell by proteolysis of larger proteins containing either polyubiquitin chains or ubiquitin fused to carboxyl extension proteins (CEPs). In humans, the two human ubiquitin-CEP genes are Uba80 and Uba52, which code for ubiquitin fused to ribosomal protein S27a and L40, respectively. Working from a recently generated physical map of human chromosome 2p16, we determined the genetic and physical location and the genomic structure of the Uba80 gene in its entirety. A comparison of Uba80 to Uba52 revealed that the two genes share a conserved 5'-end structure, but that the structure of the ubiquitin coding regions was not conserved. Analysis of 400 bp of the promoter of Uba80 revealed strong similarity not only to the Uba52 promoter, but also to the other known human ribosomal gene promoters that have been identified to date. Homology searches also detected the presence of a pseudogene for Uba80, and the structure of this sequence feature is also reported.

Amino Acid Sequence↗
PubMed1991

Voluntary control of male sexual arousal.

Forty-eight adult male volunteers attempted to suppress sexual arousal while viewing a sexually explicit videotape and generate an arousal response while viewing a neutral videotape. Attendance to the stimuli was assured by requiring subjects to provide an ongoing verbal description of the videotape they were viewing. While significant effects in controlling arousal were obtained, the degree of subject success varied as a function of the criteria used for evaluating outcome. The most conservative analysis, a simultaneous discriminant analysis procedure comparing subjects penile plethysmographic responses across the entire 3 min of each condition, revealed that in no case was a subject able to produce a pattern of sexual response that was misclassified as the condition he was attempting to emulate. Results support the value of requiring attendance to experimental stimuli and of analyzing trends/patterns of arousal across an entire experimental period in identifying attempts to "fake" sexual preferences. Theoretical and clinical implications of these findings are discussed.

Adult↗
PubMed2006

Applications of computational algorithm tools to identify functional SNPs in cytokine genes.

Understanding the functions of single nucleotide polymorphisms (SNPs) can greatly help to understand the genetics of the human phenotype variation and especially the genetic basis of human complex diseases. However, how to identify functional SNPs from a pool containing both functional and neutral SNPs is challenging. In this study, we analyzed the genetic variations that can alter the expression and function of a group of cytokine proteins using computational tools. As a result, we extracted 4552 SNPs from 45 cytokine proteins from SNPper database. Of particular interest, 828 SNPs were in the 5'UTR region, 961 SNPs were in the 3' UTR region, and 85 SNPs were non-synonymous SNPs (nsSNPs), which cause amino acid change. Evolutionary conservation analysis using the SIFT tool suggested that 8 nsSNPs may disrupt the protein function. Protein structure analysis using the PolyPhen tool suggested that 5 nsSNPs might alter protein structure. Binding motif analysis using the UTResource tool suggested that 27 SNPs in 5' or 3'UTR might change protein expression levels. Our study demonstrates the presence of naturally occurring genetic variations in the cytokine proteins that may affect their expressions and functions with possible roles in complex human disease, such as immune diseases.

Algorithms↗
PubMed2026

A genetic manipulation tool based on the GP35 recombinase for targeted gene editing in mycoplasmas of ruminants.

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78% - 100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes and obtained key insights into Mb0564, identified as a novel adhesin. The 192 to 287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.

Animals↗
PubMed2004

Experience from Lipizzan horse and salmonid species endemic to the Adriatic river system. Examples for the application of molecular markers for preservation of biodiversity and management of animal genetic resources.

Management of breeding- and free-living populations, traditionally based on phenotypic traits, relays more and more on availability of reliable information about the basic population genetic parameters as heterozygosity, mean number of alleles per locus, percentage of polymorphic loci, population structuring, genetic distances and others. Therefore, the application of molecular markers, revealing a great deal of phenotypically hidden information, becomes inevitable for population analysis. Conservation geneticists use this information for implementation of appropriate management policies. Application of molecular markers in Lipizzan horse breed, which is an example for a pedigreed breeding population, and in two endangered salmonid fish populations in Slovenia, are presented. In the Lipizzan horse breed, an insight in the population structure, overall heterozygosity, relationship between population parameters and phenotypic traits and reliability of pedigree data was gained by using molecular markers. Marble trout and Adriatic grayling were selected as examples for free-living populations, seriously endangered by human activity in the past. Development of informative molecular markers, their application and suggestions for appropriate conservation actions are described.

Animals↗
PubMed2005

Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition.

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.

Amino Acid Sequence↗
PubMed1987

The propagation of the nerve impulse.

A partial differential equation for the propagated action potential is derived using symmetry, charge conservation, and Ohm's law. Charge conservation analysis explicitly includes the gating charge when applied in the laboratory frame. When applied in the system of reference in which capacitive currents are zero, it yields a relation between orthogonal components of the ionic current allowing us to express the nonlinear ionic current in terms of the voltage-dependent membrane capacitance C(V) and the axial current that satisfies Ohm's law. The ionic current is shown to behave as C(V)V[C(V)V2]' at the foot of the action potential while the gating current behaves as C(V)V[Cg(V)V]' where Cg(V) is the capacitance associated with gating. Improved knowledge of the nonlinear current makes it possible to describe the propagated action potential in an approximated way with quasilinear partial differential equations. These equations have analytical solutions that travel with constant velocity, retain their shape, and account for other properties of the action potential. Furthermore, the quasilinear approximation is shown to be equivalent to the FitzHugh-Nagumo equation without recovery making apparent its physical content.

Action Potentials↗