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At least 1,351 records · Page 75Linked to original sources
PubMed2006

Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice.

Phosphoenolpyruvate carboxylase (PEPC), a key enzyme of primary metabolism of higher plants, is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK). Rice has three functional PPCK genes, OsPPCK1, OsPPCK2 and OsPPCK3, all of which have an intron close to the 3' end of the coding region. A novel control mechanism was found for expression of OsPPCK2, namely alternative transcription initiation, and two different transcripts were detected. The four different transcripts of the OsPPCK genes showed different expression patterns. While OsPPCK1 and OsPPCK3 were highly expressed in roots and at low levels in other organs, the two OsPPCK2 transcripts were expressed in all organs. OsPPCK3 was expressed mostly at night, while the long OsPPCK2 transcripts were present in the leaves only in the daytime. Nitrate supplementation of leaves selectively induced expression of both OsPPCK2 transcripts, while phosphate starvation only induced the shorter one. Such diverse expression patterns of OsPPCK genes suggest the importance and variety of strict activity regulation of PEPC in rice. From the correlation between gene expression and the phosphorylation level of PEPC, which was monitored as that of the maize PEPC expressed in transgenic rice plants, it was concluded that the short OsPPCK2 transcripts were expressed in rice leaf mesophyll cells upon nitrogen supplementation and phosphate starvation, whereas OsPPCK3 participated in the nocturnal phosphorylation of PEPC in these cells. Expression of PPCK proteins in rice leaves was detected by immunoblotting using a specific antiserum, and the expression of two different OsPPCK2 proteins derived from alternative transcription initiation was confirmed.

Amino Acid Sequence↗
PubMed2005

Functional analysis of six human aryl hydrocarbon receptor variants in a Japanese population.

Aryl hydrocarbon receptor (AhR) is an important transcriptional regulator involved in the induction of CYP1A1, CYP1A2, CYP1B1, UGT1A1, and UGT1A6. In this study, functional properties of four novel naturally occurring human AhR variants (K401R, N487D, I514T, and K17T/R554K) were examined along with the single variants K17T and R554K. The luciferase reporter assay using the CYP1A1 promoter reporter in HeLa cells treated with beta-naphthoflavone or 3-methylcholanthrene, which are known as typical agonists for AhR, showed that reporter activities of the K401R and N487D variants were reduced to 40 to 58% of those of wild-type (WT) but not of the other variants. Similarly, the K401R and N487D variants also reduced the omeprazole-induced reporter activities to approximately 56 and 74% of those of the WT, respectively. The reduced activities of the two variants were probably caused by the reduced protein expression levels, since the protein levels of the K401R and N487D variants were approximately 52 and 47% of the WT, respectively, without any changes in their mRNA levels. The reduced protein levels were recovered by treatment with a proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal], suggesting that the reduced protein levels were caused by the accelerated proteasomal degradation by a proteasome. Together, the current data demonstrate that the K401R and N487D variants reduce their apparent transcriptional activities, both ligand-induced and omeprazole-induced activation, probably through reduced protein expression. Thus, these two variants may influence drug metabolism through reduced induction of CYP1A1 and other target enzymes.

Genes, Reporter↗
PubMed1993

Human major histocompatibility complex class II-associated invariant chain gene promoter. Functional analysis and in vivo protein/DNA interactions of constitutive and IFN-gamma-induced expression.

Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) gene and the MHC class II molecules are physically and functionally associated within class II-expressing cell types. These genes are generally co-expressed in various cell types and coordinately induced by cytokines such as interferon-gamma (IFN-gamma). Human Ii gene shares a regulatory mechanism with MHC class II genes via the X and Y cis-acting elements. Ii X and Y are required for constitutive expression in B lymphoid cell lines and for induction by IFN-gamma in a glioblastoma cell line. The Servenius element (S) contributes 5-fold transcriptional activity to class II gene HLA-DRA, and an S homologue has been implied in Ii gene regulation. Now we report that along with X and Y, the Ii S box functions in a positive manner to regulate Ii transcription in B cell lines but not in T cell lines. In addition, S,X, and Y are all necessary for induced expression of Ii in an IFN-gamma-regulated glioblastoma cell line and in primary untransformed glial cells. Transcriptional activity of Ii X and Y elements correlates with the presence of in vivo protein/DNA interactions in Ii-expressing cells. Most interestingly, in vivo interactions are induced upon IFN-gamma induction in a time-dependent fashion. Thus, the Ii promoter contains elements that coordinately regulate expression with the MHC class II genes, and these functional sites are contacted in vivo emphasizing their importance in Ii gene transcription.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1996

A functional analysis of protooncogene Vav's role in adult human hematopoiesis.

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

Adult↗
PubMed2005

Protein kinase structure and function analysis with chemical tools.

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase-substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.

Animals↗
PubMed1999

Human papillomavirus type 6: classification of clinical isolates and functional analysis of E2 proteins.

Human papillomaviruses (HPVs) cause a variety of clinical manifestations, including the most prevalent viral sexually transmitted disease, genital warts. HPV-6 is found in a greater number of genital warts than any other HPV. To increase our understanding of the structural and functional relationships between HPV-6 isolates and to provide information for epidemiological studies, the sequences of the E2, E6 and E7 coding regions of HPV-6 genomes in clinical samples were determined. This sequence analysis was performed on isolates originally designated HPV-6a on the basis of analysis of patterns generated by restriction enzyme digestion. It was found that the designation of subtype on the basis of restriction enzyme digestion correlated poorly with the designation of subtype on the basis of sequence comparison; in fact, the clinical isolates were clearly categorized into HPV-6a and HPV-6b groups, with the previously described HPV-6vc being a member of the HPV-6a group. It was also found that the HPV-6a E2 protein is a much less potent activator of transcription than the HPV-16 E2 protein, generalizing our previous results with the HPV-6b E2 protein to this second HPV-6 E2 protein. These studies indicate that the amino acid differences observed between these natural variants of the HPV-6 E2 protein do not affect its function.

Base Sequence↗
PubMed1995

Expression and functional analysis of a baculovirus gene encoding a truncated protein kinase homolog.

Autographa californica nuclear polyhedrosis virus (AcMNPV) potentially encodes a 215-amino acid polypeptide containing 6 out of 11 motifs conserved among eukaryotic protein kinases (Morris et al., Virology 200, 360-369, 1994). We examined the expression of this gene, named pk2, at the transcriptional and translational levels and the possible role of the gene during baculovirus replication in cell culture and insect larvae. Northern (RNA) blot analysis revealed that pk2 was transcribed primarily as an early 1.2-kb RNA. Western blot analysis showed that pk2 was expressed as a 25-kDa protein, PK2, which was present both early and late during virus infection. To examine the function(s) of pk2, we constructed a mutant baculovirus, vKINdel, in which one-third of the PK2-coding region was deleted and then compared the characteristics of vKINdel with wild-type AcMNPV and a marker-rescued revertant. The pk2 deletion mutation had no discernable effect on the number, size, or appearance of plaques, the kinetics of protein synthesis or protein phosphorylation profiles during virus infection of cultured SF-21 cells. Deletion of pk2 also had no significant influence on the infectivity or virulence of the baculovirus in larval bioassays and the level of occluded virus production was normal. Thus, pk2 does not appear to have a significant influence on virus replication in the host systems examined.

Amino Acid Sequence↗
PubMed2006

Functional analysis of 11 putative essential genes in Bacillus subtilis.

Systematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these (yacA, ydiB, ydiC, ykqC, ylaN, yloQ, ymdA, yneS, yqeI, yqjK and ywlC) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes (ydiB, yloQ, yqeI and ywlC) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.

Anti-Bacterial Agents↗
PubMed2004

Structural and functional analysis of the Josephin domain of the polyglutamine protein ataxin-3.

Ataxin-3 belongs to the family of polyglutamine proteins, which are associated with nine different neurodegenerative disorders. Relatively little is known about the structural and functional properties of ataxin-3, and only recently have these aspects of the protein begun to be explored. We have performed a preliminary investigation into the conserved N-terminal domain of ataxin-3, termed Josephin. We show that Josephin is a monomeric domain which folds into a globular conformation and possesses ubiquitin protease activity. In addition, we demonstrate that the presence of the polyglutamine region of the protein does not alter the structure of the protein. However, its presence destabilizes the Josephin domain. The implications of these data in the pathogenesis of polyglutamine repeat proteins are discussed.

Ataxin-3↗
PubMed1999

Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts.

OBJECTIVE: Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function. METHODS: Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting. RESULTS: Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts. CONCLUSIONS: Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.

Actins↗
PubMed2004

New promoter mutations in the low-density lipoprotein receptor gene which induce familial hypercholesterolaemia phenotype: molecular and functional analysis.

Low-density lipoprotein receptor (LDLR) is a cell-surface glycoprotein that mediates specific uptake and catabolism of plasma LDL. Mutations located in the coding region of the LDLR gene affect the structure and function of the protein and cause familial hypercholesterolaemia (FH). Mutations in the regulatory regions of the gene are rare, but in some cases have been shown to alter the transcriptional activity of the gene and cause the FH phenotype as well. Adult heterozygous FH individuals have a markedly raised plasma cholesterol that is associated with accelerated atherosclerosis and premature coronary heart disease. The aim of this study was the functional characterization of a promoter mutation in the LDLR gene in one family from the register of Czech FH subjects. Molecular screening revealed that three members of this family carried a -27C > T nucleotide transition in the promoter sequence (calculated from the start of transcription). All three manifested a heterozygous FH phenotype. This new mutation is located between the TATA box and sterol-dependent regulatory element repeat 3. Using a luciferase reporter assay system, we analysed the transcriptional efficiency of the normal and mutant alleles. The mutation reduced promoter activity to background level. Another new promoter mutation -60C > T was identified in an unrelated patient in the conserved nucleotide sequence of the sterol-dependent regulation element repeat 2 which virtually abolished the promoter activity. We assume a causal effect of this -60C > T transition on the basis of its position in the promoter sequence.

Adolescent↗
PubMed2001

Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family.

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

Acid Phosphatase↗
PubMed2000

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide.

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.

Amino Acid Sequence↗
PubMed2000

Functional analysis of the neurofibromatosis type 2 protein by means of disease-causing point mutations.

Despite intense study of the neurofibromatosis type 2 (NF2) tumor-suppressor protein merlin, the biological properties and tumor-suppressor functions of merlin are still largely unknown. In this study, we examined the molecular activities of NF2-causing mutant merlin proteins in transfected mammalian cells, to elucidate the merlin properties that are critical for tumor-suppressor function. Most important, we found that 80% of the merlin mutants studied significantly altered cell adhesion, causing cells to detach from the substratum. This finding implies a function for merlin in regulating cell-matrix attachment, and changes in cell adhesion caused by mutant protein expression may be an initial step in the pathogenesis of NF2. In addition, five different mutations in merlin caused a significant increase in detergent solubility of merlin compared to wild type, indicating a decreased ability to interact with the cytoskeleton. Although not correlated to the cell-adhesion phenotype, four missense mutations decreased the binding of merlin to the ERM-interacting protein EBP-50, implicating this interaction in merlin inhibition of cell growth. Last, we found that some NF2 point mutations in merlin most closely resembled gain-of-function alleles in their cellular phenotype, which suggests that mutant NF2 alleles may not always act in a loss-of-function manner, as had been assumed, but may include a spectrum of allelic types with different phenotypic effects on the function of the protein. In aggregate, these cellular phenotypes provide a useful assay for identifying the functional domains and molecular partners necessary for merlin tumor-suppressor activity.

Alleles↗
PubMed1999

Functional analysis of the transcriptional coactivators p300 and CBP using ribozyme.

p300 and CERB binding protein (CBP) are transcriptional coactivator which participate many signal transduction pathways via interaction with DNA binding proteins. In this study, we investigated functions of p300 and CBP during the retinoic acid (RA) induced F9 cell differentiation using hammerhead ribozymes. In wild type F9 cells, induction of c-jun gene was observed at 18 h after treatment with RA. Interestingly, RA-dependent expression of c-jun gene was not induced in cells expressing the p300-directed ribozyme. By contrast, the CBP-directed ribozyme did not affect expression of the c-jun gene. These results suggest that p300 is critical for the induction of c-jun gene during RA-induced differentiation.

Animals↗
PubMed1993

Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation.

We cloned a cDNA coding for a putative human heme oxygenase isozyme, designated type 2 (HO-2), and analyzed its function by transient expression assays. HeLa cells transfected with either HO-2 cDNAs or a cDNA coding for authentic heme oxygenase (HO-1) expressed the activity of heme oxygenase, although no activity was detected in the mock transfected cells. Using specific anti-HO-1 antibody, we showed that expression of a HO-1 cDNA resulted in the increase in its protein levels, but HO-1 protein was not detectable in the cells expressing HO-2 cDNAs. We thus confirmed the functional identity of HO-1 and HO-2. Then, we analyzed their expression in an erythroid cell line, YN-1-0-A. Treatment with hemin or by heat shock (42 degrees C) led to a remarkable increase in the HO-1 mRNA levels, while HO-2 mRNA expression was not induced at all, suggesting that they are under separate regulation.

Cell Line↗
PubMed2005

Functional analysis of three plasmids from Lactobacillus plantarum.

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.

Arsenates↗
PubMed2007

Functional analysis of the aquaporin gene family in Caenorhabditis elegans.

Aquaporin channels facilitate the transport of water, glycerol, and other small solutes across cell membranes. The physiological roles of many aquaporins remain unclear. To better understand aquaporin function, we characterized the aquaporin gene family in the nematode Caenorhabditis elegans. Eight canonical aquaporin-encoding genes (aqp) are present in the worm genome. Expression of aqp-2, aqp-3, aqp-4, aqp-6, or aqp-7 in Xenopus oocytes increased water permeability five- to sevenfold. Glycerol permeability was increased three to sevenfold by expression of aqp-1, aqp-3, or aqp-7. Green fluorescent protein transcriptional and translational reporters demonstrated that aqp genes are expressed in numerous C. elegans cell types, including the intestine, excretory cell, and hypodermis, which play important roles in whole animal osmoregulation. To define the role of C. elegans aquaporins in osmotic homeostasis, we isolated deletion alleles for four aqp genes, aqp-2, aqp-3, aqp-4, and aqp-8, which are expressed in osmoregulatory tissues and mediate water transport. Single, double, triple, and quadruple aqp mutant animals exhibited normal survival, development, growth, fertility, and movement under normal and hypertonic culture conditions. aqp-2;aqp-3;aqp-4;aqp-8 quadruple mutants exhibited a slight defect in recovery from hypotonic stress but survived hypotonic stress as well as wild-type animals. These results suggest that C. elegans aquaporins are not essential for whole animal osmoregulation and/or that deletion of aquaporin genes activates mechanisms that compensate for loss of water channel function.

Amino Acid Sequence↗