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At least 793 records · Page 44Linked to original sources
PubMed2006

Parasitemia, anemia, and malarial anemia in infants and young children in a rural holoendemic Plasmodium falciparum transmission area.

Malarial anemia (MA) is a multifactorial disease for which the complex etiological basis is only partially defined. The association of clinical, nutritional, demographic, and socioeconomic factors with parasitemia, anemia, and MA was determined for children presenting at a hospital in a holoendemic area of Plasmodium falciparum transmission in western Kenya. Parasitemia was not associated with malaria disease severity. In univariate logistic regression, fever was significantly associated with parasitemia, and wasting was associated with increased presentation of MA. Caretaker's level of education and occupation were significantly correlated with parasitemia, anemia, and MA. Housing structure was also significantly associated with parasitemia and anemia. Bed net use was protective against parasitemia but not anemia or MA. Multivariate logistic regression models demonstrated that fever, mother's occupation, and bed net use were associated with parasitemia. In the current study, none of the factors were associated with anemia or MA in the multivariate models.

Anemia↗
PubMed2004

Cooperativity in long-range gene regulation by the lambda CI repressor.

Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (OL) located 2.4 kb from the promoter. A CI tetramer bound to OL1.OL2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant OL3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at OL3 can cooperate with a CI dimer binding to OR3. Here we show that OL3 is necessary for effective repression of PRM and that the repressor at OL3 appears to interact specifically with the repressor at OR3. The OL3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the lambda system as a powerful tool for examining long-range gene regulatory interactions in vivo.

Binding Sites↗
PubMed2004

In situ hybridization in the pathology laboratory: general principles, automation, and emerging research applications for tissue-based studies of gene expression.

Diagnostic anatomic pathologists play an important role in the care of patients through their careful evaluation of morphological features in routinely prepared histological sections stained with Hematoxylin and Eosin. Morphological assessment of tissue sections, backed by over one hundred years of experience is powerful and allows for the accurate classification and diagnosis of the majority of disease states within pathologically altered tissues. However, the appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information, which is contained within a histological section. These tissues also contain all of the cellular proteins and expressed genes, which help to ultimately determine the biological behavior of cells, as well as provide clues to the origins and pathogenesis of disease states. Technical and theoretical advances in our understanding of cellular biology have provided pathologists with powerful tools to probe beyond pure morphology into the abnormalities in both protein and gene expression that underlie human disease. These tools, which include immunohistochemistry and in situ hybridization, are playing an increasingly important role in diagnostic pathology, as well as in translational research. This review will focus on the emerging role of in situ hybridization within clinical and research laboratories, and will highlight a number of technical advances that have expanded the application of this technology.

Automation↗
PubMed2004

Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis.

The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations.

2-Acetylaminofluorene↗
PubMed2005

Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75.

Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of alpha-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Crystal structures of the Dab homology domains of mouse disabled 1 and 2.

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Complement C5a is a key mediator of meconium-induced neutrophil activation.

Meconium aspiration syndrome is a serious condition of the newborn characterized by pulmonary inflammation with substantial neutrophil infiltration. We recently showed that meconium is a potent activator of complement. The aim of the present study was to investigate a possible role for complement in meconium-induced neutrophil activation. Meconium was incubated in human whole blood anticoagulated with lepirudin, a specific thrombin inhibitor that does not affect complement activation. Complement activation was detected by measuring the terminal complement complex. Neutrophil oxidative burst and changes in CD11b and L-selectin expression were measured by flow cytometry. Complement was inhibited using the MAb 166-32 and 137-26, which block factor D and neutralize C5a, respectively. Meconium markedly activated the neutrophils, as revealed by up-regulation of CD11b, accentuation of L-selectin shedding, and induction of oxidative burst. Complement inhibition using the anti-factor D antibody completely (95-100%) blocked meconium-induced changes in CD11b and L-selectin expression, whereas oxidative burst was reduced by 60-70%. The anti-C5a antibody inhibited the neutrophil activation to the same extent as anti-factor D. The data suggest that complement activation is largely responsible for the neutrophil inflammatory responses induced by meconium in vitro and that C5a is a key mediator of this response.

Antibodies, Monoclonal↗
PubMed2003

Continuous oxygen monitoring of mammalian cell growth on space shuttle mission STS-93 with a novel radioluminescent oxygen sensor.

A compact, flow-through oxygen sensor device based on luminescence quenching was used to monitor dissolved oxygen levels during mammalian cell growth on the STS-93 mission of the Columbia space shuttle. Excitation of an oxygen-sensitive ruthenium complex was provided by a radioluminescent light source (0.9 mm in diameter, 2.5 mm long), and the intensity of the resulting luminescence was measured by a simple photodiode detector. The use of radioluminescence for the excitation light source is a unique approach that provides many features important for long-term and remote monitoring applications. For the spaceflight experiment, human lung fibroblast cells (WI-38) were grown in hollow-fiber bioreactors. Oxygen concentration was measured in the flow path both before and after the bioreactor cartridge in order to gain information about the metabolism of the cells. The sensor was found to be nonperturbing to cell growth and withstood the challenging physical conditions of shuttle launch and landing while maintaining a stable calibration function. In addition, the sensor provided physically meaningful oxygen predictions.

Aerospace Medicine↗
PubMed2005

A disulfide relay system in the intermembrane space of mitochondria that mediates protein import.

We describe here a pathway for the import of proteins into the intermembrane space (IMS) of mitochondria. Substrates of this pathway are proteins with conserved cysteine motifs, which are critical for import. After passage through the TOM channel, these proteins are covalently trapped by Mia40 via disulfide bridges. Mia40 contains cysteine residues, which are oxidized by the sulfhydryl oxidase Erv1. Depletion of Erv1 or conditions reducing Mia40 prevent protein import. We propose that Erv1 and Mia40 function as a disulfide relay system that catalyzes the import of proteins into the IMS by an oxidative folding mechanism. The existence of a disulfide exchange system in the IMS is unexpected in view of the free exchange of metabolites between IMS and cytosol via porin channels. We suggest that this process reflects the evolutionary origin of the IMS from the periplasmic space of the prokaryotic ancestors of mitochondria.

Amino Acid Motifs↗
PubMed2006

Crystal structure of a substrate complex of myo-inositol oxygenase, a di-iron oxygenase with a key role in inositol metabolism.

Altered metabolism of the inositol sugars myo-inositol (MI) and d-chiro-inositol is implicated in diabetic complications. In animals, catabolism of MI and D-chiro-inositol depends on the enzyme MI oxygenase (MIOX), which catalyzes the first committed step of the glucuronate-xylulose pathway, and is found almost exclusively in the kidneys. The crystal structure of MIOX, in complex with MI, has been determined by multiwavelength anomalous diffraction methods and refined at 2.0-A resolution (R=0.206, Rfree=0.253). The structure reveals a monomeric, single-domain protein with a mostly helical fold that is distantly related to the diverse HD domain superfamily. Five helices form the structural core and provide six ligands (four His and two Asp) for the di-iron center, in which the two iron atoms are bridged by a putative hydroxide ion and one of the Asp ligands, Asp-124. A key loop forms a lid over the MI substrate, which is coordinated in bidentate mode to one iron atom. It is proposed that this mode of iron coordination, and interaction with a key Lys residue, activate MI for bond cleavage. The structure also reveals the basis of substrate specificity and suggests routes for the development of specific MIOX inhibitors.

Animals↗
PubMed2005

Tal1/SCL binding to pericentromeric DNA represses transcription.

Tal1/SCL is a basic helix-loop-helix transcription factor critical for normal hematopoiesis. To understand the mechanisms underlying transcriptional regulation by Tal1/SCL, we combined an in vitro DNA binding strategy and an in vivo chromatin immunoprecipitation analysis to search for Tal1/SCL target regions in K562 erythroleukemia cells. A 0.4-kb genomic DNA clone containing two Tal1/SCL binding E-boxes and GATA- and SATB1-binding motifs (EEGS) was identified that localized to the pericentromeric region with high homology to satellite 2 DNA. Pericentric DNA is related to heterochromatin and gene inactivation. We found that Tal1/SCL could complex with the histone H3 lysine 9 (H3K9)-specific methyltransferase Suv39H1. Binding of Tal1/SCL to EEGS chromatin correlated with hypermethylation of H3K9 and the association of heterochromatin protein HP1 to this region. In Rep4 reporter gene assays, EEGS affected repression in a manner dependent on the expression level of Tal1/SCL that was accompanied by increased H3K9 methylation in chromatin associated with EEGS and a linked promoter. A specific histone deacetylase inhibitor, trichostatin A, relieved Tal1/SCL-mediated repression by EEGS. In addition, SATB1 bound EEGS chromatin and promoted Tal1/SCL EEGS-dependent repression. We expand the list of potential interacting partners for Tal1/SCL by demonstrating direct associations of Tal1/SCL with SATB1 and with Suv39H1. These results reveal a novel mechanism of action for Tal1/SCL and implicate heterochromatin-like silencing via a cis-acting binding motif for transcriptional repression.

Amino Acid Motifs↗
PubMed2004

Mapping immune reactivity toward Rv2653 and Rv2654: two novel low-molecular-mass antigens found specifically in the Mycobacterium tuberculosis complex.

New tools are urgently needed for the detection of latent tuberculosis (TB). We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs. Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors. However, peptides from Rv2653 were recognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule. Rv2654 also induced M. tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs. Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Animals↗
PubMed2026

Toward the clinical application of long-read sequencing in repeat-expansion disorders.

Repeat-expansion disorders (REDs) are a mechanistically and clinically well-defined subgroup of rare diseases caused by the expansion of short tandem repeats (STRs). These expansions can exceed several kilobases and show complex features, such as noncanonical secondary structures, somatic instability, repeat interruptions and allele-specific methylation. These characteristics are highly relevant for understanding disease mechanisms, clinical variability, prognosis and potentially therapeutic decision-making, but cannot be fully resolved using traditional diagnostic methods or short-read sequencing technologies. By contrast, long-read sequencing (LRS) enables accurate investigation of STR complexity in a single assay, facilitates the discovery of new pathogenic repeat expansions and drives advances in diagnostics, clinical and basic research, which may allow for better patient stratification in future clinical trials. This Perspective discusses recent LRS-driven discoveries, methodological and bioinformatic advances, and emerging diagnostic applications to illustrate the potential of LRS in reshaping both research and clinical practice.

Humans↗
PubMed2002

A brief review of recent data on some cytokine expressions at the materno-foetal interface which might challenge the classical Th1/Th2 dichotomy.

Focussing attention on cytokines at the materno-foetal interface represents one of the major advances made in the field. This owes much to the visionary views of Tom Wegmann, and to the changes brought about in the field by immunotrophism and Th1/Th2 paradigms. We review these briefly and also point out some emerging problems.However, a certain number of newly discovered cytokines do not fit into the classical Th1/Th2 dichotomy. Yet, by their capacity to activate or downregulate NK cells, by their action on adhesion molecules, and by their regulatory effects on the vascularisation process, they are of possible interest within the materno-foetal relationship. Therefore, as a first step, we have undertaken a systematic study of the expression of IL-11, IL-12, IL-13, IL-15, IL-16, IL-17 and IL-18 in the uterus, the peri-implantation embryo, and later on decidual and placental tissues throughout pregnancy. These cytokines were detected in every case, with, in each case, a precise localisation, which will be detailed, and which indeed suggests important regulatory functions, especially during implantation. In some cases, as will be shown in the peri-implantation uterus, those cells are perfectly expressed by uterine GMG-NK-like cells. Comparative ELISAs and quantitative RT-PCR have been or are being conducted, but already the expression patterns that are observed, and the very precise window of appearance that is observed for some of the GMG NK-like cells, either around or in the implanting embryo, as well as the complexity of the respective distributions, strongly suggest that, as useful as it certainly was for a while, the Th1/Th2 paradigm must now be considered as an over-simplification. Rather, the existing data point to sequential windows and are suggestive of a system where an extreme complexity is allied to very precise timing and tuning. They also suggest that the materno-foetal relationship is not simply maternal tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions governing selective immune regulation and also control of the adhesion and vascularisation processes during this dialogue.

Animals↗
PubMed2006

Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.): description of multiple novel Pax-7 splice variants.

Paired box transcription factor 7 (Pax-7) cDNA was isolated from the skeletal muscle and brain of alevin and adult stages of Atlantic salmon, identifying 10 variants categorised as novel or established insertions (ins) or deletions (del). Two putative Pax-7 paralogs were identified (denoted Pax-7alpha and Pax-7beta) on the basis of the length and sequences of intron 3 (218 and 248 bp) and versions of ins1 and ins2. Pax-7beta contained a threonine variant of ins1 (GQY[T]GPEYVYCGT), and a shortened variant of ins2 (GEAS). Pattern identification revealed the threonine variant of ins1 includes a potential phosphorylation site (casein kinase II). Thus, the tetraploid Atlantic salmon genome appears to contain at least two putative copies and multiple splice variants of Pax-7. In situ hybridisation localised Pax-7 to mononuclear cells in the fast muscle of adult Atlantic salmon, while quantitative real-time PCR showed Pax-7alpha to be more highly expressed in brain than in skeletal muscle.

Amino Acid Sequence↗
PubMed2005

Angiotensin II protects cultured midbrain dopaminergic neurons against rotenone-induced cell death.

In this study, we demonstrate that angiotensin II (Ang II) protects dopamine (DA) neurons from rotenone toxicity in vitro. Primary ventral mesencephalic (VM) cultures from E15 rats were grown for 5 days and then cultured in the presence of the mitochondrial complex I inhibitor, rotenone. Acute exposure (20 h) to 20 nM rotenone reduced the number of tyrosine hydroxylase-positive (TH+) neurons by 50 +/- 6% when compared to untreated cultures. Pre-treatment of VM cultures with 100 nM Ang II decreased TH+ neuronal loss to 25 +/- 10% at the 20-nM rotenone concentration. Ang II in the presence of the angiotensin type 1 receptor (AT1R) antagonist, losartan, was even more effective in protecting DA neurons showing a loss of only 13 +/- 4% at 20 nM rotenone. Conversely, the AT2R antagonist, PD123319, abolished the protective effects of Ang II. Furthermore, both the NMDA receptor antagonist, MK801, and the antioxidant, alpha-tocopheryl succinate (vitamin E analogue), prevented rotenone-induced toxicity. Here, we show that acute exposure of VM cultures to the pesticide rotenone leads to dopaminergic neuronal cell death and that angiotensin acting through the AT2 receptor protects dopamine neurons from rotenone toxicity.

Angiotensin II↗
PubMed2003

Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer.

In humans, the interactions between the breast cancer susceptibility protein, BRCA2, and the RAD51 recombinase are essential for DNA repair by homologous recombination (HR), failure of which can predispose to cancer. The interactions occur through conserved BRC repeat motifs, encoded in BRCA2, binding directly to RAD51. Here, we describe full and partial BRCA2 homologues from a wide range of eukaryotes, including Drosophila melanogaster and two Plasmodium species. The crystal structure of the human BRC4-RAD51 complex allows identification of residues that are important for protein-protein interaction, and defines interaction sequence fingerprints for the BRC repeat and for RAD51. These allow us to predict that most eukaryotic RAD51 and BRC repeat orthologues should be capable of mutual interactions. We find no evidence for the presence of BRC repeats in yeast, Archaea and bacteria, and their RAD51 orthologues do not fulfil the criteria for binding the BRC repeat. Similarly, human RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1, are not predicted to bind the BRC repeat. Conservation of the BRC repeat and RAD51 sequence fingerprints across a wide range of eukaryotic species substantiates the functional significance of the BRCA2-RAD51 interactions. The idea of multiple BRC repeats with binding specificity towards RAD51 leads us to suggest a possible model for the participation of BRCA2 in RAD51 nucleoprotein filament formation.

Amino Acid Sequence↗
PubMed2005

A novel locus for autosomal-dominant dilated cardiomyopathy maps to chromosome 7q22.3-31.1.

Inherited dilated cardiomyopathy (DCM) is a genetically and phenotypically very heterogeneous disease. DCM is caused by mutations in multiple genes encoding proteins that are involved in force generation, force transmission, energy production and several signalling pathways. Thus, the pathophysiology of heart failure is complex and not yet fully understood. Familial forms of DCM let the way to identify new key proteins by positional cloning and to study respective pathomechanisms that are critical for normal cardiac function, but may not have been correlated with heart disease before. Here we report a three-generation pedigree including 16 individuals affected by dilated cardiomyopathy without additional phenotypes. The pedigree is consistent with autosomal-dominant inheritance and age-related penetrance. A genome-wide linkage analysis excluded linkage to all known DCM genes and loci, whereas several close markers on chromosome 7q22.3-31.1 segregated with the disease (maximum logarithm of odds score, 4.20 at D7S471 and D7S501). The disease causing mutation lies in a 9.73 Mb interval between markers D7S2545 and D7S2554 that contains no known cytoskeletal genes. Coding exons of the candidate genes LAMB1, LAMB4 and PIK3CG were screened but no mutations were identified.

Adolescent↗