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At least 613 records · Page 34Linked to original sources
PubMed2001

Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene.

Expression of the leucocyte common antigen (CD45) in mammals is restricted to the nucleated lineages of haematopoietic cells. It appears in early progenitors in the bone marrow and is expressed at the surface of these cells throughout their differentiation. However, at least in T cells, the pattern of expression switches between different isoforms during the successive stages of differentiation in the thymus and after activation in the periphery. In order to understand the mechanisms controlling the transcription of the human CD45 gene, 2.7 kbp of the 5'-flanking region were sequenced and analysed for their ability to direct expression of a reporter gene. The only region with promoter activity was localized within the first intron of the gene. This promoter shows no tissue specificity but could be enhanced by a heterologous enhancer. Mobility shift assays showed complex but specific protein binding. The sequence in this region lacks similarity with known promoters or initiators but is highly conserved in evolution. No transcription initiation could be detected within or downstream of this region, suggesting that this might be a new type of RNA polymerase II promoter able to drive transcription from an upstream sequence. An additional exon was also found upstream of exon 1. The two exons 1 (1a and 1b) are mutually exclusive and both are spliced to exon 2. This makes the structure of the 5' region of the human CD45 gene identical to its mouse homologue.

Animals↗
PubMed2002

Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein.

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.

Amino Acid Sequence↗
PubMed1993

Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.

Binding, Competitive↗
PubMed2001

Assessment of the convergent validity of the Questions About Behavioral Function scale with analogue functional analysis and the Motivation Assessment Scale.

The present study examined the convergent validity of the Questions About Behavioral Function (QABF) scale, a behavioural checklist for assessing variables maintaining aberrant behaviour, with analogue functional analyses and the Motivation Assessment Scale (MAS). The two checklists were more highly correlated with each other than either checklist with results from the analogue sessions, and the QABF was more highly correlated with analogue sessions than the MAS. Using analogue sessions, the experimenters failed to ascertain behavioural function for a number of subjects because the behaviour problems in question were low frequency/high intensity and failed to appear during the course of the analysis, pointing out a limitation of this technology. These findings, taken together with recent research outlining the psychometric properties of the QABF, seem to support the use of the QABF in a hierarchical model of functional analysis. The implications of the findings are discussed.

Adult↗
PubMed1995

Structural and functional analysis of the promoter of the human alpha 1(XI) collagen gene.

In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.

Animals↗
PubMed2000

Preliminary functional analysis of human epidermal T cells.

The function of human epidermal T cells (ETC) is unknown. In the present study, dermal T cells (DTC), ETC and keratinocytes were cultured from normal human skin. DTC and ETC lines were expanded in medium containing interleukin 2. The autologous keratinocytes were transfected with a human papillomavirus 16 E6 and E7 plasmid to produce an immortal keratinocyte line "HEK001". Lymphocyte migration and adhesion to HEK001 was assessed in calcein fluorimetric assays. ETC migrated towards HEK001 three to four times more than DTC. ETC adhered to HEK001 two to four times more than DTC. The proportion of ETC expressing the cutaneous lymphocyte-associated antigen was greater than that of DTC (26% and 1%, respectively). The keratinocyte line HEK001 expressed ICAM-1 following stimulation with TNF-alpha or IFN-gamma and following coculture with autologous cutaneous T cells. A blocking anti-ICAM-1 antibody reduced DTC and ETC adhesion to HEK001 by 30% and 50%, respectively. Therefore, cutaneous T cells may upregulate keratinocyte ICAM-1 expression which mediates adhesion to autologous keratinocytes. These results are consistent with the hypothesis that the ETC and DTC populations are distinct. Both directed migration (epidermotropism) and selective retention may be involved in the development and maintenance of the ETC population in normal human skin.

Antigens, CD↗
PubMed2003

Common Structural Cliques: a tool for protein structure and function analysis.

Proposed is a method for locating functionally relevant atoms in protein structures and a representation of spatial arrangements of these atoms allowing for a flexible description of active sites in proteins. The search method is based on comparison of local structure features of proteins that share a common biochemical function. The method does not depend on overall similarity of structures and sequences of compared proteins or on previous knowledge about functionally relevant residues. The compared protein structures are condensed to a graph representation, with atoms as nodes and distances as edge labels. Protein graphs are then compared to extract all possible Common Structural Cliques. These cliques are merged to create Structural Templates: graphs that describe structural analogies between compared proteins. Structures of serine endopeptidases were compared in pairs using the presented algorithm with different geometrical parameters. Additionally, a Structural Template was extracted from the structures of aminotransferases, two different proteins that catalyze the same type of chemical reaction. The results presented show that the method works efficiently even in the case of large protein systems and allows for extraction of common structural features from proteins catalyzing a particular chemical reaction, but that evolved from different ancestors by convergent evolution.

Algorithms↗
PubMed1980

A semi-automatic device for pacemaker function analysis.

An external semi-automatic Pacemaker Function Analyzer (PFA) has been designed for routine examination (screening) of ambulatory-paced patients in general medical practice. The evaluation of the pacemaker (PM) function is based on recognition, decoding, and measurement of the occurrence of QRS complexes and pacing artifacts, and on logic processing of the decoded data. In this way, the state of the batteries and the integrity of the electronic circuitry and the electrodes can be determined. Other PFA applications concern supervision of hospitalized patients by interfacing the analyzer with the monitoring system of a Coronary Care Unit (CCU), transtelephone checking, and adaptation for use in specialized pacemaker clinics. The performance of the PFA was checked on 92 ambulatory patients. The PFA system recognized all but 0.29% of the QRS complexes and 0.21% of the pacing artifacts. Thirty-two of these patients were tested simultaneously by the PFA and the pacemaker clinic physician, both arriving at the same results. Twelve hospitalized patients were monitored in the CCU, after permanent PM implantation, for an average of 4 hours per patient. Although the PFA indicated 5% false-negative alarms for the hospitalized patients, it should be stressed that every true PM failure was promptly detected. It is anticipated that routine use of the PFA for management of paced patients will reduce the expenses incurred by frequent visits, as well as simplify the follow-up and surveillance of ambulatory and hospitalized patients, thereby facilitating the work of the medical staff.

Coronary Care Units↗
PubMed1984

The nurse executive role. A structural and functional analysis.

This study analyzes the structure and functions of the position of nurse executives to determine competencies required in the role, to provide a base for planning educational programs, and to identify areas for future research. These nurse executives have a dynamic role, one that has evolved as a result of role making on their part. In interviews they show a concern for meeting society's nursing needs. The interviews also show that in their positions at the executive level of the organization, they deal with complex patterns of interactional behaviors and have an ever-evolving role as the organization's power structure changes. Implications emerge for educational preparation that will help executives in their roles as nurse, administrator, and educator/researcher.

Administrative Personnel↗
PubMed2004

Isolation of p53-target genes and their functional analysis.

Mutations of the p53 gene are the most common genetic alterations found in human cancers, and are known to play crucial roles in tumor development and progression. The p53 gene encodes a protein functioning as a transcription factor, and the biological functions of p53 are manifested through the activities of its downstream genes. Identification of these downstream genes involved in the p53-signaling pathway should provide more detailed insight into the molecular mechanisms that mediate tumor-suppressor activities, as well as various responses to cellular stress. We have been attempting to isolate p53-target genes by means of various approaches, including differential display, cDNA microarray analysis, and direct cloning of the p53-binding sequences from human genomic DNA. Here I review our recent work on isolation of p53-target genes and their functional analysis. The physiological functions of p53-target genes include apoptosis (GML, p53AIP1, and STAG1), DNA repair (p53R2), inhibition of angiogenesis (BAI1), re-entry into the cell cycle (p53RFP), oxidative stress (CSR), and determination of cell fate (p53RDL1).

Animals↗
PubMed2001

[Recent advancements in MR functional analysis].

Recent advancements in magnetic resonance (MR) technology have revolutionized not only structural but also functional imaging. It is now possible to perform detailed analysis of all aspects of brain function non-invasively. From the neurological standpoint, analysis of gray matter can be categorized into (1) functional mapping and (2) neuron density measurement. Similarly, analysis of white matter can be categorized into (1) tract mapping and (2) axonal physiology. Corresponding techniques in MR imaging are functional MRI (fMRI), MR neuronography (MRN), MR tractography (MRT), and MR axonography (MRX). While fMRI is a T2* contrast based technique, the rest of the techniques are based on diffusion tensor analysis (DTA).

Brain↗
PubMed2003

Growth prediction in Class III patients using cluster and discriminant function analysis.

This longitudinal retrospective cephalometric study was undertaken in an attempt to identify subgroups of subjects with Class III malocclusions and to find discriminant functions which would help to differentiate between favourable and unfavourable growers. The material consisted of cephalometric films of 115 Class III untreated patients (59 females and 56 males, with a mean age of 11.6 +/- 1.7 and 12.7 +/- 1.3 years, respectively) who were observed for a minimum period of 1 year. All subjects were Caucasian and none could achieve an edge to edge occlusion. Hierarchical cluster analysis was used to identify Class III subgroups. Discriminant function analysis (DFA) was first applied to the whole sample and later to each of the clusters produced. Good and poor growers were identified on the basis of the change in Wits measurements with projection on the maxillary/mandibular planes bisector. The cut-off point between good and bad growers was a Wits value of 2.5 mm which was the upper limit of the 95 per cent confidence interval of measurement reproducibility. Three clinically distinguishable clusters were produced, namely long, short and intermediate facial types. The discrimination percentage (80 per cent) achieved when the DFA was performed on the whole sample was satisfactory. However, when the analysis was used on each of the clusters separately, the equation successfully predicted a good or poor outcome in 92 per cent of cluster I, in 85 per cent of cluster II and in 100 per cent of cluster III.

Analysis of Variance↗
PubMed1993

Structure-function analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130.

Here, we report the analysis of the structure-function relationship of the extracellular region of human interleukin 6 receptor (IL-6R). Upon binding of IL-6, IL-6R becomes associated extracellularly with a non-IL-6-binding but signal transducing molecule, gp130, and the IL-6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin-like domain, was responsible both for IL-6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL-6R (sIL-6R) could bind IL-6 and mediate IL-6 functions through gp130, amino acid substitutions were introduced into sIL-6R by site-directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL-6 binding have a tendency to be distributed to the hinge region between the two 'barrel'-like fibronectin type III modules and to the same side of these two 'barrels'. Amino acid residues, of which substitutions barely affected the IL-6-binding but did abolish the IL-6 signalling capability of sIL-6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL-6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL-6.

Amino Acid Sequence↗
PubMed2004

[The basics of left ventricular functional analysis with MRI and MSCT].

Over the last few years, cross-sectional imaging became clinically accepted for qualitative and quantitative analysis of left ventricular (LV) function. The results have an impact on diagnosis, therapy and follow-up, and some parameters even proved to be independent predictors of mortality. Besides well established cardiologic techniques, such as ventriculography and echocardiography, more recent radiologic techniques, especially magnetic resonance imaging (MRI) and multislice spiral computed tomography (MSCT), became accepted for imaging of LV function. Assessment of LV function with cardiac MRI is based on cine sequences. Over the last years, parallel imaging techniques, fast imaging with steady state free precession and real time sequences were successfully introduced. Further improvements were achieved by stress examinations and MR-tagging. Cardiac MSCT with retrospective ECG-gating allows for reliable determination of LV volumes. Assessment of wall motion is feasible but limited to some degree. Nevertheless, cardiac MSCT is a powerful alternative for patients with contraindications to MRI. Aim of this article is to review theoretical and technical aspects, opportunities and limitations of MRI and MSCT for functional imaging of the heart.

Adult↗
PubMed1991

The E-cadherin promoter: functional analysis of a G.C-rich region and an epithelial cell-specific palindromic regulatory element.

The cell-cell adhesion molecule E-cadherin is specifically expressed in epithelia and is involved in the maintenance of the epithelial phenotype. Expression of E-cadherin is downregulated in many poorly differentiated carcinomas, which leads to higher motility and invasiveness of the cells. To examine the mechanisms that regulate tissue-specific expression, we have characterized the promoter of the E-cadherin gene. We found that an upstream fragment (positions -178 to +92) mediates strong expression of a chloramphenicol acetyltransferase reporter gene in epithelial cells (i.e., 60% of the level obtained with simian virus 40 promoter/enhancer constructs), whereas in nonepithelial cells this promoter was either inactive or much less active. By DNase I footprinting and gel retardation analysis as well as through functional dissection of the regulatory sequences, we identified two regions that contribute to tissue-specific activity of the promoter: (i) a G-C-rich region between -25 and -58 that generates basic epithelial promoter activity, most likely in combination with an "initiator" element present at the single transcription start site of the gene, and (ii) a palindromic sequence between -75 and -86 (named E-pal) that potentiates the activity of the proximal E-cadherin promoter and confers epithelial cell-specific activity on a simian virus 40 promoter. The E-pal sequence is homologous to cis regulatory elements active in keratin gene promoters and competes with these elements for nuclear factor binding. Interestingly, the activity of the E-cadherin promoter was reduced in dedifferentiated breast carcinoma cells, indicating that the identified elements are subject to negative regulation during tumor progression.

Animals↗
PubMed2004

Functional analysis of the transcriptional activator XlnR from Aspergillus niger.

The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XlnR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal DNA-binding domain has been determined experimentally. Deletion mutants of the xlnR gene were constructed to localize the functional regions of the protein. The results showed that a putative C-terminal coiled-coil region is involved in nuclear import of XlnR. After deletion of the C-terminus, including the coiled-coil region, XlnR was found in the cytoplasm, while deletion of the C-terminus downstream of the coiled-coil region resulted in nuclear import of XlnR. The latter mutant also showed increased xylanase activity, indicating the presence of a region with an inhibitory function in XlnR-controlled transcription. Previous findings had already shown that a mutation in the XlnR C-terminal region resulted in transcription of the structural genes under non-inducing conditions. A regulatory model of XlnR is presented in which the C-terminus responds to repressing signals, resulting in an inactive state of the protein.

Active Transport, Cell Nucleus↗
PubMed1984

Functional analysis of human T cell subsets defined by monoclonal antibodies. VI. Distinct and opposing immunoregulatory functions within the OKT8+ population.

In the present study, we investigated the immunoregulatory potential of OKT8+ cells after in vitro activation. Initial studies had demonstrated that the T cell marker OKT4 identifies T cell sets containing helper cells, whereas OKT8 reacts with the suppressor and cytotoxic T cell effectors. More detailed analysis of the OKT4+ subset, however, demonstrated that there is functional heterogeneity in the OKT4+ population. The evidence for this heterogeneity arose from studies on (1) the relative radiosensitivities of the distinct immunoregulatory functions of cells contained within this set, and, (2) the effects of the state of activation of this population on immune function. For example, OKT4+ cells included radiosensitive helper cells, radioresistant helper cells, and radiosensitive inducers of suppressor cells. Furthermore, after activation, the OKT4+ population also contained cells capable of suppressing B cell differentiation. Since activation of the OKT4+ population results in the emergence of cells with counterbalancing immunoregulatory properties, it was of interest to determine whether the state of activation of the OKT8+ population influences the immunoregulatory potential of this subset. In the experiments reported here E+ cells were cultured with pokeweed mitogen (PWM) for 60-70 h and then thoroughly depleted of OKT4+ cells, leaving OKT8+ cells. The ability of the PWM-activated OKT8+ cells (first culture) to exert suppressive activity was determined by adding graded numbers of these cells to fresh autologous OKT4+ cells and B cells. The cell mixtures were cultured in the presence of PWM for 5 days and then assayed for plaque-forming-cell (PFC) activity by the reverse hemolytic plaque assay. Our studies demonstrate that activation of OKT8+ cells results in the emergence of cells with apparently counterbalancing immunoregulatory properties. Activated nonirradiated OKT8+ cells consistently suppressed B cell immunoglobulin production. However, the immunoregulatory function mediated by irradiated activated OKT8+ cells is highly dependent on the magnitude of the helper activity obtained with fresh OKT4+ cells. Thus, irradiated activated OKT8+ cells suppressed the generation of PFC only when the level of helper activity was optimal. However, when the level of help was not optimal, no suppressor activity was observed; rather, under these conditions, irradiated activated OKT8+ cells amplify the PFC response. We would emphasize that the amplification function of irradiated OKT8+ cells depends strictly on the presence of fresh OKT4+ cells. Irradiated activated OKT8+ cells alone are not helper cells, since addition of these cells to B cells thoroughly dep

Antibodies, Monoclonal↗