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At least 1,405 records · Page 78Linked to original sources
PubMed2006

Relationship between salt-bridge identity and 14-helix stability of beta3-peptides in aqueous buffer.

We report a systematic analysis of the relationship between salt bridge composition and 14-helix structure within a family of model beta-peptides in aqueous buffer. We find an inverse relationship between side-chain length and the extent of 14-helix structure as judged by CD. Introduction of a stabilizing salt bridge pair within a previously reported beta-peptide ligand for hDM2 led to changes in structure that were detectable by NMR.

Buffers↗
PubMed2006

Detecting differential and correlated protein expression in label-free shotgun proteomics.

Recent studies have revealed a relationship between protein abundance and sampling statistics, such as sequence coverage, peptide count, and spectral count, in label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics. The use of sampling statistics offers a promising method of measuring relative protein abundance and detecting differentially expressed or coexpressed proteins. We performed a systematic analysis of various approaches to quantifying differential protein expression in eukaryotic Saccharomyces cerevisiae and prokaryotic Rhodopseudomonas palustris label-free LC-MS/MS data. First, we showed that, among three sampling statistics, the spectral count has the highest technical reproducibility, followed by the less-reproducible peptide count and relatively nonreproducible sequence coverage. Second, we used spectral count statistics to measure differential protein expression in pairwise experiments using five statistical tests: Fisher's exact test, G-test, AC test, t-test, and LPE test. Given the S. cerevisiae data set with spiked proteins as a benchmark and the false positive rate as a metric, our evaluation suggested that the Fisher's exact test, G-test, and AC test can be used when the number of replications is limited (one or two), whereas the t-test is useful with three or more replicates available. Third, we generalized the G-test to increase the sensitivity of detecting differential protein expression under multiple experimental conditions. Out of 1622 identified R. palustris proteins in the LC-MS/MS experiment, the generalized G-test detected 1119 differentially expressed proteins under six growth conditions. Finally, we studied correlated expression of these 1119 proteins by analyzing pairwise expression correlations and by delineating protein clusters according to expression patterns. Through pairwise expression correlation analysis, we demonstrated that proteins co-located in the same operon were much more strongly coexpressed than those from different operons. Combining cluster analysis with existing protein functional annotations, we identified six protein clusters with known biological significance. In summary, the proposed generalized G-test using spectral count sampling statistics is a viable methodology for robust quantification of relative protein abundance and for sensitive detection of biologically significant differential protein expression under multiple experimental conditions in label-free shotgun proteomics.

Bacterial Proteins↗
PubMed2006

Study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach.

Increased serum haptoglobin concentration and changes in its glycosylation have been reported in certain cancer types. Information for hepatocellular carcinoma (HCC) has not yet been available. In this study, we aimed to carry out a systematic analysis of serum concentrations of haptoglobin (Hp) and its glycoforms in the patients with HCC and noncancer patients only with chronic liver diseases (CLD) and to examine their clinical values. This study was divided into two major parts, (1) measurement of serum Hp concentration, and investigation of its value in the diagnosis of HCC, and (2) quantitative analysis of Hp glycoforms with alpha-2,6-sialylation and/or alpha-1,6-fucosylation by using lectin affinity purification and 2D gel electrophoresis and investigation of their relationships with tumor stage. The concentrations of serum Hp in HCC patients were significantly higher than those in noncancer patients with CLD. With the use of serum concentrations of Hp and alpha-fetoprotein, a logistic regression (LR) model was developed from the training data set and used to classify the validation cases. At a specificity of 95%, the sensitivity for HCC detection was 79%. Comparing serum concentrations of alpha-2,6-sialylated Hp (S-Hp) and alpha-1,6-fucosylated Hp (F-Hp) between HCC and CLD patients suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. 2D gel analysis of S-Hp and F-Hp identified a total of 18 glycoforms. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species was found in the HCC patients. Serum concentrations of these glycoproteins were significantly higher in the patients with advanced tumors, suggesting their tumor-specific nature. We have shown that serum Hp is a potential biomarker in the diagnosis of HCC. The combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation is specific to HCC and associated tumor progression.

Adult↗
PubMed1999

Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression.

During seed maturation, the transcriptional activity of napin genes is regulated by developmental signals involving the transcriptional activator ABI3 and abscisic acid (ABA). To localize cis elements involved in the seed-specific activity of the napin napA promoter, a systematic analysis was performed focusing on two major element complexes, the B-box and RY/G. Substitution mutation analysis using promoter-reporter gene fusions in stable transgenic tobacco showed synergistic interactions between elements within these complexes. The distal part of the B-box shows similarities to abscisic acid response elements and the proximal portion contains a CA-rich element. In vitro studies involving Exonuclease III protection and electrophoretic mobility shift assays revealed binding by nuclear proteins to elements within the B-box. The distal and proximal parts of the B-box were found to bind distinct nuclear protein complexes. By gain-of-function analysis with a tetramer of the B-box fused to a truncated (-46) cauliflower mosaic virus (CaMV) 35S minimal promoter, it was demonstrated that the B-box mediates strong activity in seeds. Further, it was shown that the elements in the B-box constitute an ABA-responsive complex, since the B-box tetramer mediates ABA-responsiveness in vegetative tissues to a construct containing the CaMV virus 35S enhancer (-343 to -90). Thus, the seed-specific activity of the napA promoter relies on the combinatorial interaction between the RY/G complex and the B-box ABA-responsive complex during the ABA response in seed development.

2S Albumins, Plant↗
PubMed2000

Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2.

The Arabidopsis Genome Initiative has released up to now more than 80% of the genome sequence of Arabidopsis thaliana. About 70% of the identified genes have at least one paralogue. In order to understand the biological function of individual genes, it is essential to study the structure, expression and organization of the entire multigene family. A systematic analysis of multigene families, made possible by the amount of genomic sequence data available, provides important clues for the understanding of genome evolution and plasticity. In this paper, four multigene families of A. thaliana are characterized, namely LCAD, HD-GL2, LGT and MYST. Members of HD-GL2 and LCAD have already been reported in plants. The LGT genes specify proteins containing motifs of glycosyl transferase. No plant genes similar to the LGT genes have been reported to date. The novel MYST family, most likely plant-specific, encodes proteins with no identified function. Sequencing and in silico analysis led to the characterization of 29 novel genes belonging to these four gene families. The organization, structure and evolution of all the members of the four families are discussed, as well as their chromosome location. Expression data of some of the paralogues of each family are also presented.

Alcohol Oxidoreductases↗
PubMed1998

Recent advances in the molecular genetics of cancer. Second joint conference of the American Association of Cancer Research and the European Association of Cancer Research, Oxford, 9-12 September 1997.

The second joint conference of the AACR and the EACR held in Oxford from 9-12 September 1997 was successful from many vantage points. While providing an optimal setting in which European and American cancer researchers could meet and exchange information, the conference had an excellent scientific programme which encompassed both methodological updates on important models used in cancer research and presentations of recent key advances in the molecular genetics of cancer. Lower eukaryotes are established model organisms used to elucidate fundamental but complex eukaryotic processes, such as those involved in tumorigenesis and cancer progression, and the progressive availability of their genome sequence makes them even more attractive. Transgenic mouse models are increasingly used not only for the study of one gene of interest but for investigation of the interactions among genes involved in the same pathway. The family of tumour suppressor genes is growing fast and several presentations were devoted to recently identified members such as the Von Hippel-Lindau gene, the FHIT gene and the PTEN gene. The systematic analysis of loss of heterozygosity on multiple loci in tumour specimens can provide the basis for preliminary models of molecular multistep progression in some tumour types, even though this is limited by the high degree of complexity found. Mechanisms of cell cycle regulation and apoptosis continue to be dissected and to constitute a fruitful area of investigation, with important recent insights on the p53-MDM2 autoregulatory loop and on the involvement of E2F-1 in apoptosis.

Animals↗
PubMed1998

Steps towards a theory of medical practice.

This article has a threefold intention. 1. It intends to contribute to the clarification of the question in what respect medicine may be called a science and in what respect a practice. 2. It proposes a concept of clinical methodology (including clinical-ethical aspects), as a theory of medical practice that is one component of theoretical medicine. 3. It sketches an approach and some steps towards a systematic analysis of medical-clinical practice. In the first part, the position that medicine is a "practical science" is criticized. It is shown that the supposed opposition of "theoretical" and "practical" sciences is misconstrued. The relevant distinctions are those between knowledge, research, and scientific practice. In the second part, and analysis of the structure and function of descriptive and normative data and of clinical knowledge and principles in clinical practice is given, and a strategy for solving the deep problems of clinical methodology and ethics is sketched.

Decision Making↗
PubMed2002

Insights from mouse models of absence epilepsy into Ca2+ channel physiology and disease etiology.

1. Changes in intracellular Ca2+ ([Ca2+]i) levels provide signals that allow neurons to respond to a host of external stimuli. A major mechanism for elevating [Ca2+]i is the influx of extracellular Ca2+ through voltage-gated channels (Ca(V)) in the plasma membrane. Malfunction in Ca(V) due to mutations in genes encoding channel proteins are increasingly being implicated in causing disease conditions, termed channelopathies. 2. Seven spontaneous mutations with cerebellar ataxia and generalized absence epilepsy have been identified in mice (tottering, leaner, rolling Nagoya, rocker, lethargic, ducky, and stargazer), and these overlapping phenotypes are directly related to mutations in genes encoding the four separate subunits that together form the multimeric neuronal Ca(V) complex. 3. The discovery and systematic analysis of these animal models is helping to clarify how different mutations affect channel function and how altered channel function produces disease.

Animals↗
PubMed1999

Fragments of functional proteins: role in endocrine regulation.

Systematic analysis of structures, localization, formation and biological activities of endogenous peptides derived from functional proteins, such as hemoglobin, myelin basic protein, immunoglobulins, etc., allowed establishing the basic features of that group of compounds. The sets of these peptides in mammalian tissues, or "tissue-specific peptide pools" are: (i) tissue specific; (ii) stable at normal conditions; (iii) conservative in the same tissues of different mammalian species; (iv) dependent on the general state of homeostasis of tissue or the whole organism. Formation of such peptides has features of both conformation and site specificity and also involves the action of carboxy- and amino-peptidases. As a result, the families of structurally related families of peptides are generated. The fragments of functional proteins exhibit a wide range of the biological effects, characteristic both for hormones and parahormones, from hormone-releasing to growth-regulatory activity. At the same time, the molecular mechanisms of action of the majority of such peptides are unknown. On the basis of the data obtained the components of tissue-specific peptide pools are considered to form a novel regulatory system, complementary to other peptidergic systems such as hormonal, nervous, immune, etc. The biological role of the fragments of functional proteins in vivo and the patterns of interaction with other regulatory systems are suggested.

Animals↗
PubMed1999

The screening and diagnosis of autistic spectrum disorders.

The Child Neurology Society and American Academy of Neurology recently proposed to formulate Practice Parameters for the Diagnosis and Evaluation of Autism for their memberships. This endeavor was expanded to include representatives from nine professional organizations and four parent organizations, with liaisons from the National Institutes of Health. This document was written by this multidisciplinary Consensus Panel after systematic analysis of over 2,500 relevant scientific articles in the literature. The Panel concluded that appropriate diagnosis of autism requires a dual-level approach: (a) routine developmental surveillance, and (b) diagnosis and evaluation of autism. Specific detailed recommendations for each level have been established in this document, which are intended to improve the rate of early suspicion and diagnosis of, and therefore early intervention for, autism.

Asperger Syndrome↗
PubMed2004

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells.

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.

Aerobiosis↗
PubMed2004

SiRNA based strategies for inhibition of apoptotic pathways in vivo--analytical and therapeutic implications.

Apoptosis is a genetically controlled mechanism of cell death which is important for embryogenesis, metamorphosis, tissue homeostasis and tumor regression of multicellular organisms. In normal cells as well as in transformed cells signals released from the cytoplasm and/or the cell membrane can trigger the activation of caspases which in turn cleave many cellular substrates, leading to the characteristic morphology of apoptosis. Systematic analysis and dissection of apoptotic pathways was obtained by the use of knock out or transgenic organisms, expressing dominant active or negative proteins. The use of antisense oligonucleotides (ASO) for analysis of apoptotic pathways was commonly restricted to cultured cells, because of the low efficacy of ASO in in vivo experiments. In order to investigate the contribution of specific apoptotic pathways in the onset and maintenance of disease in vivo experiments are needed. This approach allows the analysis of apoptotic pathways within their physiological/pathophysiological environment. The combination of recent advances in in vivo gene delivery with siRNA technique for efficient gene silencing provides new, unique possibilities to study apoptotic pathways thereby evaluating new molecular therapeutic strategies in vivo. In this minireview we will focus on the use of RNA interference for analytical and therapeutical suppression of apoptotic pathways in vivo with special consideration of the liver.

Animals↗
PubMed2002

Construction and evaluation of a manikin for perioperative heat exchange.

BACKGROUND: During surgery hypothermia can be avoided only if the heat exchange between the body surface and the environment can be controlled. To allow a systematic analysis of this heat exchange, we constructed and evaluated a copper manikin of the human body. METHODS: The manikin consists of six tubes (head, trunk, two arms and two legs) painted matt-black to simulate the emissivity of the human skin. Hot-water mattresses are bonded to the inner surface of the copper tubes to set the surface temperature. Calibrated heat flux transducers were placed on the following points to determine the heat exchange coefficient for radiation and convection (hRC) of the manikin: Forehead, chest, abdomen, upper arm, forearm, dorsal hand, anterior thigh, anterior leg and foot. Room temperature was set to 22 degrees C. Surface temperature of the manikin was set between 22 degrees C and 38 degrees C. The hRC was determined by linear regression analysis as the slope of the temperature gradient between the manikin and the room versus the measured heat flux. Subsequently we studied five minimally clothed volunteers in a climate chamber. Initial chamber temperature was set to 29 degrees C and was lowered slowly to 12 degrees C. The hRC was determined as described above for each volunteer. RESULTS: The hRC of the manikin was 11.0 W m(-2) degrees C(-1) and hRC of the volunteers was 10.8 W m(-2) degrees C(-1). CONCLUSION: The excellent correlation of hRC between the volunteers and the manikin will allow the manikin to be used for standardised studies of perioperative heat exchange.

Adult↗
PubMed2003

The time course of spoken word learning and recognition: studies with artificial lexicons.

The time course of spoken word recognition depends largely on the frequencies of a word and its competitors, or neighbors (similar-sounding words). However, variability in natural lexicons makes systematic analysis of frequency and neighbor similarity difficult. Artificial lexicons were used to achieve precise control over word frequency and phonological similarity. Eye tracking provided time course measures of lexical activation and competition (during spoken instructions to perform visually guided tasks) both during and after word learning, as a function of word frequency, neighbor type, and neighbor frequency. Apparent shifts from holistic to incremental competitor effects were observed in adults and neural network simulations, suggesting such shifts reflect general properties of learning rather than changes in the nature of lexical representations.

Artificial Intelligence↗
PubMedDate not supplied

Age-impaired impulse flow from nucleus basalis to cortex.

Recent studies have renewed interest in the role of acetylcholine (ACh) in the cognitive changes associated with ageing and dementia. Deficits in cortical choline acetyltransferase (ChAT) in Alzheimer's disease have been consistently demonstrated, while other research has suggested a connection between deterioration of cortical ACh fibres and dementia. However, despite clear biochemical and anatomical evidence for a fall in ACh in dementia, results of therapeutic trials with cholinergic agonists, precursors and cholinesterase inhibitors have been inconsistent. Such findings suggest that cortical cholinergic disorders are not wholly a function of simple biochemical change; alterations of impulse flow along cholinergic fibres could well be as debilitating. An important extrinsic source of cortical ACh innervation derives from neurones diffusely located in rat basal forebrain, denoted the nucleus basalis (NB). We have now investigated the impulse conduction properties of cortically projecting, putatively cholinergic NB axons in adult and aged rats and have found that conduction latencies from NB to frontal cortex are significantly longer (by 51%) in aged animals. In addition, systematic analysis varying cortical stimulation depth revealed that these longer latencies are due entirely to decreased conduction velocities in the subcortical fibre projections. Indeed, intracortical velocities were virtually identical in the two groups. Our results indicate that ageing occasions a decrease in the temporal fidelity of impulse flow in the cholinergic input to the cortex from the NB, a previously overlooked but potentially important element in cognitive deficits that occur with age.

Aging↗
PubMedDate not supplied

AIDS virus-specific cytotoxic T lymphocytes in lung disorders.

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.

Cytotoxicity, Immunologic↗
PubMed2000

Production of hepatitis B surface antigen in transgenic plants for oral immunization.

Here we present data showing oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) in preclinical animal trials. Mice fed transgenic HBsAg potato tubers showed a primary immune response (increases in HBsAg-specific serum antibody) that could be greatly boosted by intraperitoneal delivery of a single subimmunogenic dose of commercial HBsAg vaccine, indicating that plants expressing HBsAg in edible tissues may be a new means for oral hepatitis B immunization. However, attainment of such a goal will require higher HBsAg expression than was observed for the potatoes used in this study. We conducted a systematic analysis of factors influencing the accumulation of HBsAg in transgenic potato, including 5' and 3' flanking elements and protein targeting within plant cells. The most striking improvements resulted from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells.

Administration, Oral↗
PubMed2001

Transcriptional regulation and function during the human cell cycle.

We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle-dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle-regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

Apoptosis↗