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At least 1,279 records · Page 71Linked to original sources
PubMed2003

Functional analysis of CFTR chloride channel activity in cells with elevated MDR1 expression.

Using the patch-clamp method, we investigated a relationship between MDR1 expression and its effects on the CFTR channel function. Incubation of CaCo-2 cells with increasing concentrations of doxorubicin resulted in a reduction of CFTR chloride channel activity in a dose-dependent manner. This reduction was associated with a decrease of CFTR mRNA and simultaneous up-regulation of MDR1 mRNA in the presence of doxorubicin. Similar alteration of the CFTR function was observed in CaCo-2 cells transiently overexpressing MDR1. No alterations of the cAMP-dependent chloride currents were observed in COS-1 cells transiently co-expressing CFTR and MDR1 from strong CMV promoters. This indicated that repression of CFTR by MDR1 induction requires the presence of the native CFTR promoter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter.

The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFN-stimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-l) element. The KCS-l element is similar to the 15-bp KCS element so far unique to the human and mouse RNA-dependent PKR kinase gene promoters. The KCS element of the PKR kinase (PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-l element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-l element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-l-binding proteins shared some properties with PKR KCS-binding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-l element revealed that the ADAR1 KCS-l element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-l element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-l element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter.

Adenosine Deaminase↗
PubMed2003

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation.

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.

Amino Acid Sequence↗
PubMed1991

Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.

Animals↗
PubMed1995

Functional analysis of the Bacillus subtilis purT gene encoding formate-dependent glycinamide ribonucleotide transformylase.

The purT gene from Bacillus subtilis encoding the formate-dependent glycinamide ribonucleotide transformylase T was cloned by functional complementation of an Escherichia coli purN purT double mutant. The nucleotide sequence revealed an open reading frame of 384 amino acids. The purT amino acid sequence showed similarity to the enzyme phosphoribosylaminoimidazole carboxylase encoded by the purK gene but not to the N10-formyltetrahydrofolate-dependent glycinamide ribonucleotide transformylase N enzyme encoded by the purN gene. The glycinamide ribonucleotide transformylase T level was repressed in cells grown in rich medium compared to minimal-medium-grown cells. However, when the culture entered the stationary-growth phase the enzyme level increased in rich medium and decreased in minimal medium. By comparing the deduced amino acid sequence of the B. subtilis purT gene product with translated nucleotide sequences in various databanks, evidence for the existence of putative purT genes in the Gram-negative bacteria Pasteurella haemolytica and Pseudomonas aeruginosa was obtained.

Acyltransferases↗
PubMed2006

Isolation and functional analysis of luxS in Serratia plymuthica RVH1.

Autoinducer-2 (AI-2) is a signalling molecule proposed to act as a 'universal' signal for interspecies communication, regulating niche-specific genes with diverse functions in various bacteria. We have previously reported the production of AI-2 in strains from different Serratia species and recently, the luxS gene of two Serratia strains was isolated and shown to affect distinct phenotypes. In the current work, we have identified the luxS gene of Serratia plymuthica RVH1, and studied its effect on the N-acyl-L-homoserine lactone-regulated 2,3-butanediol fermentation, production of extracellular protease, chitinase, nuclease and antibacterial compound, and on biofilm formation. None of these phenotypes was affected by luxS knockout. However, overexpression of the luxS gene in RVH1, which was accompanied by an increased production of AI-2, resulted in a slower growth. This growth retardation was also observed when RVH1 was grown in spent culture medium from the AI-2-overproducing strain, but not in spent culture medium from the luxS knockout strain. These results suggest that luxS primarily fulfils a metabolic role and may not regulate a coordinated behaviour controlled by cell-to-cell signalling in S. plymuthica RVH1.

4-Butyrolactone↗
PubMed2000

Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor.

The trefoil peptide intestinal trefoil factor (ITF) plays a critical role in the protection of colonic mucosa and is essential to restitution after epithelial damage. These functional properties are accomplished through coordinated promotion of cell migration and inhibition of apoptosis. ITF contains a unique three-looped trefoil motif formed by intrachain disulfide bonds among six conserved cysteine residues, which is thought to contribute to its marked protease resistance. ITF also has a seventh cysteine residue, which permits homodimer formation. A series of cysteine-to-serine substitutions and a C-terminally truncated ITF were made by PCR site-directed mutagenesis. Any alteration of the trefoil motif or truncation resulted in loss of protease resistance. However, neither an intact trefoil domain nor dimerization was required to promote cell migration. This pro-restitution activity correlated with the ability of the ITF mutants to activate mitogen-activated protein (MAP) kinase independent of phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, only intact ITF retained both phosphatidylinositol 3-kinase and the EGF receptor-dependent antiapoptotic effect in HCT116 and IEC-6 cells. The inability to block apoptosis correlated with a loss of trefoil peptide-induced transactivation of the EGF receptor or Akt kinase in HT-29 cells. In addition to defining structural requirements for the functional properties of ITF, these findings demonstrate that distinct intracellular signaling pathways mediate the effects of ITF on cell migration and apoptosis.

Amino Acid Sequence↗
PubMed2001

Phenotypic and functional analysis of tumor-infiltrating lymphocytes compared with tumor-associated lymphocytes from ascitic fluid and peripheral blood lymphocytes in patients with advanced ovarian cancer.

To investigate and compare the phenotype and function of lymphocytes collected from patients harboring advanced ovarian cancer, leukocytes from peripheral blood (n = 18), ascitic fluid (n = 13) and tumor tissues (n = 13) were evaluated for the relative proportions of lymphocyte subsets, including CD3+, CD4+, CD8+, CD19+, CD56 and the early (CD25) and late (HLA-DR) activation markers on CD3+ T cells. The ability to synthesize type 1 cytokines (IFN-gamma and IL-2) and a type 2 cytokine (IL-4) was assessed by flow cytometry. In all patients, T cells (CD3+) were the major leukocyte population detected in each tissue, with CD4+ T cells being dominant in peripheral blood lymphocytes (PBL) and tumor-associated lymphocytes (TAL) but not in tumor-infiltrating lymphocytes (TIL) (CD4:CD8 ratios: 3.0 vs. 2.0 vs. 1.0, respectively). CD19+ lymphocytes (B cells) and CD56+ lymphocytes (NK cells) were significantly higher in PBL compared to TAL and TIL (p < 0.05). TAL and TIL had a higher proportion of T cells expressing the late activation marker HLA-DR compared to PBL. In contrast, no significant differences were detected in PBL, TAL and TIL in the expression of the early activation marker CD25. Type 1 cytokines were the dominant type produced by in vitro stimulated T cells for each population, with a greater proportion of IFN-gamma+ T cells in TAL and TIL compared to PBL (p < 0.01), and a higher proportion of IL-2+ T cells in PBL compared with TAL and TIL (p < 0.05). Low percentages of IL-4+ T cells (i.e. Th2) were detected in each tissue. Taken together, these data demonstrate the recruitment and accumulation of high concentrations of antigen-experienced T lymphocytes in TAL and TIL compared to PBL. However, low surface expression of IL-2 receptor (i.e. CD25), as well as depressed intracellular IL-2 production in chronically stimulated TAL and TIL suggests that the impaired antitumor function commonly detected in these lymphocyte populations may be secondary to an acquired dysregulation of the IL-2 pathway.

Adult↗
PubMed2004

Structure-function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus.

Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp null-mutant Drosophila: In the living animal, mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37 degrees C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.

Amino Acid Sequence↗
PubMed1987

[Traumatic scapho-lunate dissociation. Functional analysis, surgical therapy and results].

The traumatic scapho-lunate dissociation represents a heavy lesion of the carpal ligaments. The instability and incongruity of the joint causes a considerable loss of the function of the hand. The mechanical situation of the proximal carpal row is demonstrated on the base of anatomical investigations and the mechanism of the accident in a subluxation-rotation injury is shown. The therapeutic consequences are pointed out and the author's own operations procedure (reconstruction of ligaments) with results is presented.

Carpal Bones↗
PubMed1976

[Scintigraphic functional analysis with the gamma camera during renal perfusion (author's transl)].

The controlled progress of transplanted kidneys has already been useful. We have succeeded in finding a simple method to study the function of the renal parenchyma and the collecting system. This method was also applied to determine a flow volume relation during perfusion in comparison to impulse rates. In contrast the conventional parameters, which only describe a partial function of the organ, this simple method will deliver comprehensive information before transplantation.

Animals↗
PubMed1997

Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment.

For an understanding of the aberrant biology seen in mouse mutations and identification of more subtle phenotype variation, there is a need for a full clinical and pathological characterization of the animals. Although there has been some use of sophisticated techniques, the majority of behavioral and functional analyses in mice have been qualitative rather than quantitative in nature. There is, however, no comprehensive routine screening and testing protocol designed to identify and characterize phenotype variation or disorders associated with the mouse genome. We have developed the SHIRPA procedure to characterize the phenotype of mice in three stages. The primary screen utilizes standard methods to provide a behavioral and functional profile by observational assessment. The secondary screen involves a comprehensive behavioral assessment battery and pathological analysis. These protocols provide the framework for a general phenotype assessment that is suitable for a wide range of applications, including the characterization of spontaneous and induced mutants, the analysis of transgenic and gene-targeted phenotypes, and the definition of variation between strains. The tertiary screening stage described is tailored to the assessment of existing or potential models of neurological disease, as well as the assessment of phenotypic variability that may be the result of unknown genetic influences. SHIRPA utilizes standardized protocols for behavioral and functional assessment that provide a sensitive measure for quantifying phenotype expression in the mouse. These paradigms can be refined to test the function of specific neural pathways, which will, in turn, contribute to a greater understanding of neurological disorders.

Animals↗
PubMed2005

[Phenotypic and functional analysis of T lymphocytes in cancer patients].

In preclinical tumor model and in human cancer, tumor antigen specific T lymphocytes play a key role in the control of tumor development. Nevertheless in numerous cases, the infiltrating tumor T cells do not seem to influence the clinical progression of the tumor. A better phenotypic and functional characterization of T cells in close contact with tumor associated with a comprehensive analysis of tumor evasion mechanism to the host response should lead to an optimization of cancer immunotherapy protocols.

Antigens, CD↗
PubMed1997

Functional analysis of the promoter for the human CYP1B1 gene.

Our laboratory has cloned the cDNA (Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W. , and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099) and gene (Tang, Y. M., Wo, Y.-Y. P., Jabs, E. W., Stewart, J. C., Sutter, T. R., and Greenlee, W. F. (1996) J. Biol. Chem. 271, 28324-28330) for human CYP1B1, a new member of the cytochrome P450 superfamily. Here, we report on the mapping and function of the CYP1B1 promoter. The CYP1B1 promoter is fully functional, when it is uncoupled from upstream enhancer elements. Deletion analysis and site-directed mutagenesis identified four regulatory elements required for maximum promoter activity: two antisense Sp1 sites (-84 to -89 and -68 to -73), a TATA-like box (-34 to -29), and an initiator motif (-5 to +3). The initiator and the TATA-like elements are both required for basal promoter activity, with enhanced activity mediated by the two antisense Sp1 elements. The CYP1B1 initiator was demonstrated by in vitro transcription analysis to be a positioning element that maintained fidelity of transcription from a single site. Specific binding to a CYP1B1 initiator probe by human nuclear extract proteins was competed either by the highly homologous murine terminal deoxynucleotidyl transferase initiator or, to a lesser extent, by the adenovirus major late initiator. Taken together, these results indicate that the structure and function of the CYP1B1 promoter confers constitutive expression of the gene and assures fidelity of transcription initiation from a single site. The CYP1B1 promoter is distinct from the promoters of the closely related cytochrome P450s CYP1A1 and CYP1A2 and is structurally and functionally similar to the promoters of constitutively expressed genes and at least two viruses.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Functional analysis of mRNA scavenger decapping enzymes.

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.

Base Pairing↗
PubMed1996

Functional analysis of cemented medial unicompartmental knee arthroplasty.

Gait analysis was used to study patients who underwent cemented medial unicompartmental knee arthroplasty (UKA) for medial compartment degenerative knee disease. All had excellent clinical function of the operated knee, with minimal involvement of other joints. Ten patients met the inclusion criteria for this study. Gait findings from this study group, as well as those from a similar cohort of patients who had undergone total knee arthroplasty (TKA), were compared with those for a group of normal patients. A greater percentage of patients (7 of 10) following UKA maintained the normal biphasic flexion/extension moment pattern about the knee or demonstrated a quadriceps overuse pattern, when compared with similar groups following TKA. In addition, the adduction moment in patients following UKA was significantly larger than in patients following TKA. The postoperative limb alignment correlated with the peak adduction moment recorded during the patient's gait analysis. The postoperative alignment may explain the differences in the adduction moment between the UKA and TKA patients, as there tended to be residual varus in the UKA population. The results of this study imply that preservation of the anterior cruciate ligament during UKA allows patients to maintain normal quadriceps mechanics, and that residual varus alignment subjects a medial UKA to higher loads.

Aged↗
PubMed2000

Phenotypic and functional analysis of bovine gammadelta lymphocytes.

The studies have provided the first comprehensive comparison of the factors regulating activation and proliferation of WC1+ and WC1- gammadelta T cells. The investigation has shown that accessory molecules essential for activation and function of WC1+ and WC1- gammadelta T cells and the sources and roles of cytokines in activation of gammadelta T cells through the T cell receptor (TCR). The study has also shown that the role of cytokines in activation and function of gammadelta T cells activated indirectly through cytokines secreted by ab T cells, accessory cells and antigen presenting cells (APC). Cytokines were differentially produced by subpopulations of gammadelta T cells under different conditions of activation. The investigation obtained in this study has revealed that factors account for activation and proliferation of gammadelta T cells in cultures designed to study MHC-restricted responses to antigens. Evidence obtained here has shown there is biological relevance to activation under these culture conditions that points to potential regulatory and effector functions of gammadelta T cells. The investigations have also provided the information needed to begin identifying and characterizing antigens recognized by the TCR repertoires of WC1+ and WC1- gammadelta T cells. Finally, the investigations have provided the information needed to begin analysis of the mechanisms by which gammadelta T cells modulate MHC restricted immune responses to pathogens and derived vaccines.

Animals↗