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Results for “functional experiments”

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At least 325 records · Page 18Linked to original sources
PubMed2001

The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.

In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.

Alleles↗
PubMed1999

H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.

Adenocarcinoma↗
PubMed2005

Dorsoventral patterning of the Xenopus eye: a collaboration of Retinoid, Hedgehog and FGF receptor signaling.

In the developing spinal cord and telencephalon, ventral patterning involves the interplay of Hedgehog (Hh), Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling. In the eye, ventral specification involves Hh signaling, but the roles of RA and FGF signaling are less clear. By overexpression assays in Xenopus embryos, we found that both RA and FGF receptor (FGFR) signaling ventralize the eye, by expanding optic stalk and ventral retina, and repressing dorsal retina character. Co-overexpression experiments show that RA and FGFR can collaborate with Hh signaling and reinforce its ventralizing activity. In loss-of-function experiments, a strong eye dorsalization was observed after triple inhibition of Hh, RA and FGFR signaling, while weaker effects were obtained by inhibiting only one or two of these pathways. These results suggest that the ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling. We argue that similar mechanisms might control ventral neural patterning throughout the central nervous system.

Animals↗
PubMed2006

IAN family critically regulates survival and development of T lymphocytes.

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell-receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.

Animals↗
PubMed1990

On the cost functions for the control of the human arm movement.

The aim of our investigation is to understand the mechanisms which control the movement of the human arm. The arm is here considered as a redundant system: the shoulder, elbow and wrist joints, which provide three degrees of freedom, combine to move the hand in a horizontal plane, i.e. a two dimensional space. Thus the system has one extra degree of freedom. Earlier investigations of the static situation led to the hypothesis that independent cost functions were attached to each of the three joints and that the configuration chosen for a given target position is that which provides the minimum total cost (Cruse 1986). The aim of the current investigation was to look for measurable values corresponding to the hypothetical cost functions. Experiments using pointers of different lengths attached to the hand showed that the strategy in choosing the joint angles are independent of the limb length. The muscle force necessary to reach a given angle is increased by a spring mounted across a joint. In this situation the angles of the loaded joint are changed for a given target point to give way to the force effect. This leads to the conclusion that the hypothetical cost functions are not independent of the physiological costs necessary to hold the joint at a given angle. The cost functions seem to depend on joint angle and on the force which is necessary to hold the joint in a given position. Cost functions are measured by psychophysical methods. The results show U-shaped curves which can be approximated by parabolas. The position of minimum cost (maximum comfort) for one joint showed no or weak dependency on the angles of the other joints. For each subject these "psychophysical" cost functions are compared with the hypothetical cost functions. The comparison showed reasonable agreement. This supports the assumption that the psychophysically measured "comfort functions" provide a measure for the hypothetical cost functions postulated to explain the targeting movements. Targeting experiments using a four joint arm which has two extra degrees of freedom showed a much larger scatter compared to the three joint arm. Nevertheless, the results still conform to the hypothesis that also in this case the minimum cost principle is applied to solve the redundancy problem. As the cost function for the whole arm shows a large minimum valley, quite a large range of arm positions is possible of about equal total costs.(ABSTRACT TRUNCATED AT 400 WORDS)

Arm↗
PubMed1997

High resolution magnetic resonance imaging application in anatomy: the extensor digitorum muscle insertion on the first phalanx.

Classical dissection may give unsatisfactory results because of the presence of artifacts due to both the embalming process and displacement of the anatomical structures. This spatial disturbance could explain the divergent descriptions found in the literature about the presence, or the absence, of an insertion of the extensor digitorum muscle (ED) at the first phalanx (P1). Preliminary experiments by Van Sint Jan et al. (1996) found the same contradiction: dissections did not show a real tendon attachment, whereas a functional experiment seemed to show that "something" should exist between ED and P1 to explain the results. This paper presents the results of an in vitro MRI study of this anatomical area. A 7-T NMR microscope was used to collect accurate, noninvasive data. Subsequently, surface rendering was performed to visualize the structures in a three-dimensional manner. The results of this MRI study, together with functional data obtained in an earlier study, showed that no real insertion of ED on P1 exists. However, some collagenic fibers were occasionally-observed running from the ventral aspect of ED to both P1 and the metacarpo-phalangeal joint capsule. Those few collagenic fibers would play a secondary role in the extension of P1.

Collagen↗
PubMed1999

Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus.

The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb ultraviolet light (UV) and putative functions in plants include a role as UV protectants. In support of this possible function we demonstrate here that UV light induces accumulation of several TIAs as well as expression of the Tdc gene in C. roseus. In addition, in tobacco a Tdc-gusA construct was found to be specifically induced by UV-B light. Lack of induction by UV-A or other wavelengths of light indicate that Tdc expression is regulated by a specific UV-B receptor and corresponding signal transduction pathway. To identify UV-responsive Tdc promoter elements, a loss-of-function analysis was performed, in which deletion derivatives were fused to the gusA reporter gene and analysed in transgenic tobacco plants. Truncation of the Tdc promoter from -1818 (relative to the start of transcription) to -160 reduced expression levels two-fold without affecting the qualitative UV response. Deletion to -37 further reduced expression levels five-fold, but the delta37 promoter also remained UV-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which the transcriptional activities of tetramerized subfragments fused to truncated promoters were analysed. Combination of the data identified several functional regions in the -160 to +198 promoter. The - 160 to -99 region acts as the main transcriptional enhancer. UV-responsive elements appeared to be redundant in the -160 Tdc promoter and to reside between -99 and -37 and between -37 and + 198.

Alkaloids↗
PubMed2004

RNAi in a postmodern, postgenomic era.

As dsRNA-mediated interference (RNAi) libraries become more widely available, genome-wide loss of function experiments in cell culture are likely to become a routine laboratory tool. RNAi screens can then be conducted to test gene functions in different assays in a variety of cell types and across species. In comparing these large phenotypic data sets, researchers will gain a new perspective, enabling them to see each gene as part of a fluid network that is remodelled in response to changes in environmental and developmental context, and with the passage of evolutionary time.

Gene Library↗
PubMed2004

Functional mapping of the Fc gamma RII binding site on human IgG1 by synthetic peptides.

Receptors specific for the Fc part of IgG (Fc gamma R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc gamma R may result in autoimmunity. Thus, the modulation of IgG-Fc gamma R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc gamma RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P(234) and S(298) were synthesized and used in binding and functional experiments. Binding of the peptides to Fc gamma R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T(256)-P(271), which bound to soluble recombinant Fc gamma RIIb with K(d)=6 x 10(6) M(-1). The biotinylated peptides R(255)-P(271) and T(256)-P(271) complexed by avidin exhibited functional activity; they induced Fc gamma RIIb-mediated inhibition of the BCR-triggered Ca(2+) response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF-alpha and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc gamma RII-binding part of IgG1.

Binding Sites↗
PubMed2006

Role of delivery and trafficking of delta-opioid peptide receptors in opioid analgesia and tolerance.

Changes in the number of receptors on the cell surface lead to modulations of physiological functions and pharmacological responses of neurons. Recent studies show that delta-opioid peptide (DOP) and mu-opioid peptide (MOP) receptors have distinct subcellular localizations in neurons. In nociceptive small neurons in the dorsal root ganglia, DOP receptors are sorted into neuropeptide-containing secretory vesicles, enabling the stimulus-induced cell surface expression of these receptors. MOP receptors are constitutively expressed on the cell surface. The physical interaction between DOP receptors and MOP receptors seems to be an important mechanism for the modulation of receptor functions. Experiments in animals show that MOP-receptor-mediated spinal analgesia is enhanced and morphine tolerance does not develop when DOP receptor functions are pharmacologically or genetically attenuated. Thus, the delivery and trafficking of DOP receptors are crucial processes that modulate opioid analgesia and tolerance.

Analgesia↗
PubMed1999

Functional improvement of elderly residents of institutions.

Studies have shown that some elderly persons who suffer decline in activities of daily living (ADL) functioning experience an improvement. This phenomenon has been examined mainly among elderly persons in the community using summary ADL indices. This article examines functional improvement among 2,527 residents of institutions for semi-independent and frail elders in Israel in four specific ADLs--bathing, eating, bladder continence, and mobility--at two points in time, 2 to 4 years apart. Demographic, functional, and institutional variables were used to predict functional improvement through logistic regression. The variables were found to differentially affect each ADL, highlighting two opposite aspects of institutionalization--deterioration, on the one hand, and rehabilitation through intervention by highly trained staff, on the other.

Activities of Daily Living↗
PubMedDate not supplied

[Experimental study of the effect of sodium nitrate on the functional neutrophil and macrophage activity].

The impact of sodium nitrite on functional activity of macrophages and neutrophiles of the abdominal cavity and blood of mice BALB/c was investigated in vitro and in vivo experiments. Functional activity was estimated according to formation of oxygen active forms. It was shown that sodium nitrite significantly inhibits formation of reactive oxygen species released from mouse peritoneal neutrophiles and macrophages, when they are being activated in vitro. Sodium nitrite was given in water at concentration 500.0 mg/l mice for 210 days immunodepression action was revealed which expressed reduction by leukopoes and decrease functional activity of blood neutrophils. Under the same conditions nitrite did not effect on functional activity of resident peritoneal macrophages.

Animals↗
PubMed2004

Comparative anatomy of nitrergic intrinsic choroidal neurons (ICN) in various avian species.

Intrinsic choroidal neurons (ICN) represent a peculiar feature of eyes in higher primates and birds. They account for up to 2000 in human and duck eyes but are virtually absent or rare in all other mammalian species investigated so far. It has been suggested that ICN are involved in regulation of ocular blood supply, hence influencing intraocular pressure, and changes in choroidal thickness, thus influencing accommodation. The present study was undertaken in order to compare differences in various avian species with respect to ICN as well as to provide data on some avian species relevant for experimental ophthalmic research, i.e. chicken and quail. Choroids from 12 avian species were processed for NADPH-diaphorase histochemistry or, in some cases, neuronal nitric oxide synthase immunocytochemistry. ICN were quantified and normalized to mean choroidal area. Three choroids of each galliformes (i.e. chicken, quail, turkey) and anseriformes (i.e. Muscovy duck, Mallard duck, goose) were rastered in squares of 1 mm2 and x/y coordinates were transferred into a 3D-diagram with the amount of ICN represented in the z-axis. ICN were detected in all species investigated. They were predominantly small cells with soma diameters of 20-30 microm. In turkey, and to a lesser amount in chicken, a subpopulation of ICN with somal diameters of up to 70 microm was observed. Highest mean cell counts were found in goose (6195.4; turkey 3558.4; chicken 1681.4; Muscovy duck 785.4; Mallard duck 640.8; quail 440.2). Normalized to choroidal area, highest mean cell counts were (per mm2): 12.62 in goose, 4.42 in both chicken and turkey, 2.86 in quail, 2.66 in Mallard duck and 1.89 in Muscovy duck. In galliformes, ICN were found to be accumulated temporo-cranial, while in anseriformes they were arranged in a more belt-like fashion, passing from cranio-nasal to temporo-caudal. Our results show that besides Muscovy duck, other avian species appear as suitable models for further functional experiments on ICN. The temporo-cranial accumulation of ICN in galliformes and the belt-like arrangement in anseriformes may reflect special functional requirements in regions of high visual acuity.

Anatomy, Comparative↗
PubMed1996

Racial and ethnic disparities in self-assessed health status: evidence from the National Survey of Families and Households.

We examined racial and ethnic disparities in global health assessment and functional limitations of daily activities among whites, blacks and Hispanics, and within the Hispanic origin among Mexicans, Puerto Ricans, Cubans, and 'Others'. Logistic regression were employed to estimate the log odds of reporting 'poor health' and 'having functional limitations' among 12,814 respondents from the 1987-1988 National Survey of Families and Households. Compared with whites, blacks had an increased risk of reporting poor health and functional limitations. Hispanics had even a higher risk of reporting poor health, but did not have an increased risk of reporting functional limitations. Among Hispanics, Mexicans were more likely than whites to report poor health, whereas Puerto Ricans were more likely than whites to experience functional limitations. Both race and ethnicity remain important factors in explaining the disparities in self-assessed health status independent of socioeconomic status (SES). Meanwhile, the way self-assessed health status varies with ethnicity is importantly stratified by SES as measured by income and education. These results suggest that future research should analyze the interplay between ethnicity and SES rather than assuming measuring either captures all the important variation.

Activities of Daily Living↗
PubMed1988

Correlation of immunoregulatory function with cell phenotype in cord blood lymphocytes.

The strong suppressor activity of cord T lymphocytes contrasts markedly with their mainly CD4 (helper) rather than CD8 (suppressor) phenotype. We studied the phenotype of cord CD3, CD4 and CD8 cells compared to adult cells using the monoclonal antibodies, 2H4, 4B4, and UCHL1. Almost all cord CD4 lymphocytes carried the suppressor-inducer marker 2H4, whereas 4B4+ UCHL1+ helper-inducer cells were virtually absent; CD8 cord cells were also of the 2H4+ 4B4- UCHL1- phenotype. In contrast in adult peripheral blood, half of the T cells, whether CD4 or CD8, were 2H4+ and half 4B4/UCHL1+. The suppressor-inducer phenotype of cord T cells was shown, in parallel functional experiments, to correlate with their enhanced proliferation to lectin and poor production of immunoglobulin and with the ability of cord mononuclear cells to suppress proliferation and immunoglobulin production by adult cells in co-culture experiments. These results indicate that the major imbalance in the cord CD4 subset in favour of 2H4 cells can explain many of the functional differences from adult cells. However, involvement of other cell types, in particular of the monocyte lineage, is necessary to explain other properties of immunocompetent cord cells.

Adult↗
PubMed2006

The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity.

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.

Animals↗
PubMed1999

Elicitor-responsive promoter regions in the tryptophan decarboxylase gene from Catharanthus roseus.

The tryptophan decarboxylase (Tdc) gene from Catharanthus roseus (Madagascar periwinkle) encodes a key enzyme in biosynthesis of terpenoid indole alkaloids. The expression of the Tdc gene is transcriptionally induced by fungal elicitors. Tdc upstream sequences from -1818 to +198 relative to the transcriptional start site were functionally analysed to identify cis-acting elements that determine basal expression or respond to elicitor. In a loss-of-function analysis promoter derivatives with 5' or internal deletions fused to the gusA reporter gene were analysed in transgenic tobacco plants. Whereas promoter activity dropped considerably following deletion down to -160, this short promoter derivative was still elicitor-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which subfragments were tested as tetramers cloned on two different truncated promoters. Combination of the data resulted in the identification of three functional regions in the -160 promoter. The region between -160 to -99 was shown to act as the main transcriptional enhancer. Two separable elicitor-responsive elements were found to reside between -99 and -87 and between -87 and -37. These two elements are not redundant in the Tdc promoter, since their combination gave a distinct elicitor response.

Amino Acid Sequence↗
PubMed1998

Promoters of nuclear-encoded respiratory chain complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression.

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCl) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5' upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the beta-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the -200/-100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCl promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.

Arabidopsis↗