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At least 1,765 records · Page 98Linked to original sources
PubMed2003

Functional analysis of murine uterine natural killer cells genetically devoid of oestrogen receptors.

Uterine Natural Killer (uNK) cell differentiation in vivo requires oestrogen (E) priming prior to progesterone (P). Hybridomas between uNK precursor and SP2/0 cells express message for E receptor (ER)alpha but nor PR. However, mature, rodent and human uNK cells lack these receptors. To functionally assess requirements for uNK cell expression of ERalpha or ERbeta during precursor differentiation, marrow was transplanted from either ERalpha(o/o) (alphaERKO) or ERbeta(o/o) (betaERKO) mice into alymphoid RAG-2(o/o)/gammac(o/o) females. Recipients were mated and their implantation sites were examined by light microscopy, morphometry and ultrastructure. High numbers of uNK cells were established from each donor strain. Graft-derived uNK cells were similar in number and morphology to uNK cells of normal mice, suggesting that neither alpha- nor beta-ER is required for uNK precursor cell differentiation. Induction of spiral artery modification in the transplant recipients indicated that graft-derived uNK cells had functional properties. A novel technique for rapid isolation of highly purified uNK cells from normal mice using Dolichos biflorus agglutinin (DBA) lectin-conjugated magnetic beads was employed to obtain RNA. Expression of alpha- and beta-ER was absent by RT-PCR from NK cells isolated from the uterus, supporting the conclusions from the in vivo study.

Animals↗
PubMed2005

Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites.

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional dystrophin proteins and potential conversion of severe DMD into milder Becker muscular dystrophy (BMD) phenotypes. We have previously described 37 exon-internal AONs that induce skipping of 14 DMD exons in human control myotube cultures. Here, we report 77 new AONs, effectively targeting an additional 21 exons. Of the 114 AONs thus far tested, 72 (67%) were effective. AON design initially was based on a partial overlap with predicted open secondary structures in the target RNA. We have analyzed various AON and target exon parameters in retrospect. Interestingly, we observed significantly higher SF2/ASF, SC35, and SRp40 values (as predicted by ESEfinder) for effective AONs when compared with ineffective AONs. In addition, the distance to the 3' splice site was significantly smaller for effective AONs. No other significant correlations were observed. Our results suggest that effective exon-internal AONs primarily act by blocking SR binding sites (which often correspond to open structures) and that ESEfinder may be used to refine AON design for DMD and other genes.

Base Sequence↗
PubMed1989

Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements.

In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells.

Amino Acid Sequence↗
PubMed2004

Identification, functional analysis and expression in a heterotopic heart transplant model of CXCL9 in the rat.

CXCR3 chemokines are of particular interest because of their potential involvement in a variety of inflammatory diseases, including the rejection of organ transplants. Although the rat is one of the most appropriate animals for using to study transplantation biology, the structural and functional characteristics of CXCL9 [monokine induced by interferon-gamma (Mig)] in this experimental model have not been described. Therefore, we recently conducted a series of experiments to identify and characterize the rat CXCL9 gene. Accordingly, we isolated rat CXCL9 cDNA and genomic DNA. The rat CXCL9 gene encodes a protein of 125 amino acids and spans a 3.5 kbp DNA segment containing four exons in the protein-coding region. We then analysed mRNA expression in various tissues. Transcripts for the gene were found to be expressed at high levels in the lymph nodes and spleen. Then, to confirm the function of the identified gene, rat CXCL9 was transiently expressed in COS-1 cells. Rat recombinant Mig displayed chemotactic properties and induced CXCR3 internalization in CD4+ T cells. Lastly, we analysed the expression of rat CXCL9 in a heterotopic heart allograft model. Both mRNA and protein levels of intragraft CXCL9 were significantly increased following transplantation of ACI to LEW hearts when compared with syngeneic controls. These findings indicate that rat CXCL9 has an in vivo role in the infiltration of CD4+ T cells in the transplanted graft.

Amino Acid Sequence↗
PubMed1995

Transfer function analysis of vagal control of heart rate during synchronized vagal stimulation.

Synchronized electrical stimulation was used to study the heart rate (HR) response to fluctuations in parasympathetic input to the sinus node in anesthetized dogs. This was obtained by varying the time interval (interpulse interval) between stimulatory vagal pulses. Spectral methods were used to estimate transfer functions between the excitatory signal and the resulting HR response for different intensities of vagal stimulation. The intensity of vagal stimulation was proportional to the number of pulses delivered in each cardiac cycle. From the estimated transfer functions, and based on a mathematical model of the time course of ACh concentration at the sinus node, filter models were derived by using a system identification approach. HR response was characterized by a combination of two different filter behaviors: a low-pass filter behavior of mean cut-off frequency of 0.065 Hz and an all-pass filter behavior. The magnitude of the low-pass filter gain decreased with increasing intensity of vagal stimulation. The magnitude of the all-pass filter gain increased and then decreased with increasing intensity of vagal stimulation. The all-pass filter characteristics of HR response during synchronized stimulation of the vagus nerves are specific to this mode of stimulation, because they were not observed in nonsynchronized modes of vagal stimulation. We can conclude that, during synchronized vagal stimulation, the HR response exhibits both a slow dynamic component and a fast component related to beat-to-beat variations.

Animals↗
PubMed1997

In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE).

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, we have mutated the Hoxa-1 3'RARE using the Cre-loxP targeting strategy, and studied its functional role during mouse development. We find that this enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, we also find that the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Interestingly, although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5.

Animals↗
PubMed2001

Functional analysis of the iron(II) etiocorrphycene incorporated in the myoglobin heme pocket.

The iron(III) complex of 2,7,12,17-tetraethyl-3,6,11,18-tetra-methylcorrphycene, an isomeric heme, was complexed with apomyoglobin to examine the ligand binding ability of the novel macrocycle under physiological conditions. The reconstituted holoprotein was found to be functionally active at pH 7.4 and 20 degrees C and to bind oxygen and carbon monoxide reversibly with a half-saturation pressure at 6.7 and 3.5mmHg, respectively. Equilibrium affinities for these ligands are one to two orders of magnitude lower than those reported for native myoglobin. The functional anomaly was ascribed to the geometric and electronic strain on the iron(II) atom in the trapezoidal coordination core of corrphycene.

Heme↗
PubMed2004

Wire transfer function analysis for castellated dual-energy x-ray detectors.

An investigation into the spatial resolving power of a castellated linear dual-energy x-ray detector array is reported. The detector was developed for use in aviation security screening applications. Experiments employing different gauges of lead wire are used to plot a wire transfer function. A numerical simulation is developed to predict and underpin the empirical results. The suitable processing of the castellated detector signals helps to maintain spatial resolving power while affording a 50% reduction in x-ray sensing elements. This encouraging result has formed the basis for an ongoing investigation into materials discrimination capability of the castellated detector array.

Journal Article↗
PubMed1999

Functional analysis of the Ume3p/ Srb11p-RNA polymerase II holoenzyme interaction.

The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis. This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery. This report describes two domains required for Ume3p-RNA Pol II holoenzyme association. One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding. The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box. In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo. Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity. In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo. Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.

Adenosine Triphosphatases↗
PubMed1998

Functional analysis of domains in the Byr2 kinase.

The activation of mitogen-activated protein (MAP) kinase cascades by the Ras GTPase is an evolutionarily conserved signal transduction mechanism. To better understand the interaction between Ras and its target kinase, we study the yeast Schizosaccharomyces pombe where the Ras1 GTPase activates the Byr2 kinase. The Byr2 kinase contains an N-terminal regulatory region and a C-terminal kinase region. The regulatory region can be divided into a sterile-alpha motif (SAM) that binds Ste4, a Ras1-binding domain (RBD) that binds activated Ras1, and a catalytic binding domain (CBD) that interacts with the Byr2 kinase domain. To analyze the importance of functional domains of the Byr2 kinase, a biological assay was used that exploited the ability of Byr2 to partially bypass the need for Ras1 in sporulation. Analysis of mutants using this assay showed that SAM and RBD were very important for Ras1-stimulated sporulation. Three activating mutations were identified within the N-terminal lobe of the Byr2 kinase domain that partially bypassed the need for Ras1 for sporulation. These activating mutations may identify a region of the Byr2 kinase domain that interacts with the CBD since mutations in the CBD which disrupt binding to the kinase domain also increase Byr2 function.

Fungal Proteins↗
PubMed2005

Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming.

Most aquatic invertebrates adapt to environmental osmotic changes primarily by the cellular osmoconforming process, in which osmolytes accumulated in their cells play an essential role. Taurine is one of the most widely utilized osmolytes and the most abundant in many molluscs. Here, we report the structure, function and expression of the taurine transporter in the Mediterranean blue mussel (muTAUT), as a key molecule in the cellular osmoconforming process. Deduced amino acid sequence identity among muTAUT and vertebrate taurine transporters is lower (47-51%) than that among vertebrate taurine transporters (>78%). muTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration than vertebrate taurine transporters. This seems to reflect the internal environment of the mussel; higher NaCl and taurine concentrations. In addition to the hyperosmotic induction that has been reported for cloned taurine transporters, the increase in muTAUT mRNA was unexpectedly observed under hypoosmolality, which was depressed by the addition of taurine to ambient seawater. In view of the decrease in taurine content in mussel tissue under conditions of hypoosmolality reported previously, our results lead to the conclusion that muTAUT does not respond directly to hypoosmolality, but to the consequent decrease in taurine content. By immunohistochemistry, intensive expression of muTAUT was observed in the gill and epithelium of the mantle, which were directly exposed to intensive osmotic changes of ambient seawater.

Amino Acid Sequence↗
PubMed2026

Identification and functional analysis of MeJA-responsive bHLH family genes in Taraxacum kok-saghyz.

Taraxacum kok-saghyz (T. kok-saghyz) is considered a highly promising alternative source of natural rubber (NR), as its roots synthesize high-molecular-weight NR comparable to that produced by Hevea brasiliensis. The basic helix-loop-helix (bHLH) family of transcription factors (TFs) plays crucial roles in plant organogenesis, hormonal signal transduction, and the regulation of secondary metabolism. This study aimed to systematically identify TkbHLH family members and to elucidate their potential functions in responding to methyl jasmonate (MeJA) and regulating root development. Based on the T. kok-saghyz genome, 172 TkbHLH members were identified and phylogenetically classified into 16 subfamilies. Among these, 37 genes were selected due to their significant induction by MeJA. Sequence analysis confirmed all encoded proteins contain the conserved bHLH domain. Subcellular localization verified nuclear localization of five core TkbHLH proteins. Interactions were shown by yeast two-hybrid and bimolecular fluorescence complementation, revealing these proteins form homodimers and heterodimers. Notably, a specific interaction was detected between TkbHLH162 and TkHMGS1, a key enzyme in the mevalonate (MVA) pathway, suggesting a potential molecular link between JA signaling and the rubber biosynthesis precursor pathway. Functional characterization via overexpression assays showed that selected TkbHLH genes significantly either promoted or inhibited root elongation. In summary, this study presents the first systematic characterization of the bHLH TF family in T. kok-saghyz, elucidating its involvement in JA signal response, protein interaction networks, and root development regulation. These findings provide a crucial foundation for further investigation into the molecular mechanisms by which TkbHLH TFs influence root morphogenesis and NR biosynthesis in T. kok-saghyz.

Taraxacum kok-saghyz (T. kok-saghyz)↗
PubMed2004

Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis.

A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekeeping genes whose function can be analyzed by disruption in the chicken DT40 B-cell line.

Animals↗
PubMed2003

Functional analysis of PCCB mutations causing propionic acidemia based on expression studies in deficient human skin fibroblasts.

Propionic acidemia (PA) is a recessive disorder caused by a deficiency of propionyl-CoA carboxylase (PCC), a dodecameric enzyme composed of two different proteins alpha-PCC and beta-PCC, nuclear encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause PA and to date, up to 47 different allelic variations in the PCCB gene have been identified in different populations. In this work, we describe the expression studies of 18 PCCB sequence changes in order to elucidate their functional consequences. We have used a PCCB-deficient transformed fibroblast cell line to target the wild-type and mutant proteins to their physiological situation, analysing the effect of the mutations on PCC activity and protein stability. Of the 18 mutant proteins tested for activity, those carrying the L17M and A497V substitutions showed an activity similar to the wild-type one, which proves that these changes do not have any effect on protein activity. The other 16 mutant proteins exhibited two different functional behaviours, 3 retained substantial activity (K218R, R410W and N536D), and the remaining 13 proteins showed null or very low activity. Western blot analysis demonstrated instability only for the L519P, R512C and G112D mutant proteins. We have proved the pathogenicity of R67S, R165Q and G112D mutation in PCCB gene, expressed for the first time in this work. The information derived from the expression analysis is discussed in the phenotype and genotype context in order to improve the knowledge of this complex disease.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa.

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.

Amino Acid Sequence↗
PubMed1995

Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization.

Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted alpha-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.

Amino Acid Sequence↗
PubMed1980

Genetic and functional analysis of tryptophan transport in Malpighian tubules of Drosophila.

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w+ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.

Animals↗
PubMed1995

Functional analysis of the Drosophila CDC2 Dm gene in fission yeast.

The cdc2+ gene product (p34cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeast Schizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34cdc2 proteins, we have constructed a S. pombe strain in which the yeast cdc2+ gene has been replaced by its Drosophila homologue CDC2Dm (the CDC2Dm strain). This CDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34CDC2Dm recognizes all the essential S. pombe cdc2+ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56cdc13-117, suggesting that this S. pombe cyclin might interact less efficiently with the Drosophila protein than with its native p34cdc2 counterpart. This CDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34CDC2Dm (and/or that p34cdc2-independent pathways are used). The CDC2Dm gene produces a 'wee' phenotype, and it is largely insensitive to the action of the S. pombe wee1+ mitotic inhibitor, suggesting that Drosophila wee1+ homologue might not be functionally conserved. This CDC2Dm strain is hypersensitive to UV irradiation, to the same degree as wee1-deficient mutants. A strain which co-expresses the Drosophila and yeast cdc2+ genes shows a dominant wee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms.

Animals↗