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At least 469 records · Page 26Linked to original sources
PubMed1987

Strontium-89 radionuclide therapy: a dosimetric study using impulse response function analysis.

In a series of patients receiving 89Sr palliation for metastasised prostatic carcinoma, strontium renal plasma clearance was found to vary from 0.14 to 11.81 day-1, and the extent of skeletal metastatic disease seen on 99Tcm-MDP images varied from a few small metastases to a superscan. Using a numerical technique based on impulse response function (IRF) analysis, we have investigated the effect of such variation between patients on 89Sr dosimetry. The whole-body IRF, HWB(t), is defined by the deconvolution of the whole-body strontium retention function, RWB(t), with the plasma retention function, P(t). For patients with minimal metastatic bone disease we assumed HWB(t) = HO(t), where HO is the IRF derived from the International Commission on Radiological Protection model for normal strontium metabolism. The strontium plasma clearance, k, was allowed to vary, and the resulting variation of RWB(t), P(t) and absorbed dose to bone marrow calculated. By convoluting P(t,k) with the IRF measured for a discrete metastasis, the effect of varying k on tumour dose was investigated. Tumour and bone marrow dose were shown to change by a factor of three as k varied over the range observed in patients. For patients with extensive metastatic bone disease we assumed HWB(t) = (1-beta)HO(t) + beta HS(t), where HS was the IRF measured for a superscan patient and beta was a parameter reflecting the extent of skeletal metastatic disease. The effect of varying beta on tumour and bone marrow dose was investigated, and dose shown to decrease by a factor of five as beta increased from zero to unity. Impulse response function analysis was found to be a powerful and useful aid in clarifying the relationship between strontium kinetics and 89Sr dosimetry.

Bone Marrow↗
PubMed2004

Probing the S100 protein family through genomic and functional analysis.

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family.

Animals↗
PubMed2006

Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain.

Nonribosomal peptide synthetases are modular proteins that operate in an assembly line fashion to bind, modify, and link amino acids. In the E. coli enterobactin NRPS system, the EntE adenylation domain catalyzes the transfer of a molecule of 2,3-dihydroxybenzoic acid to the pantetheine cofactor of EntB. We present here the crystal structure of the EntB protein that contains an N-terminal isochorismate lyase domain that functions in the synthesis of 2,3-dihydroxybenzoate and a C-terminal carrier protein domain. Functional analysis showed that the EntB-EntE interaction was surprisingly tolerant of a number of point mutations on the surface of EntB and EntE. Mutational studies on EntE support our previous hypothesis that members of the adenylate-forming family of enzymes adopt two distinct conformations to catalyze the two-step reactions.

Amino Acid Sequence↗
PubMed1987

Functional analysis of a retroviral host-range mutant: altered long terminal repeat sequences allow expression in embryonal carcinoma cells.

A retroviral host-range neomycin-resistant myeloproliferative sarcoma virus mutant, which is expressed in the embryonal carcinoma cell lines F9 and PCC4aza1R, was molecularly cloned and analyzed. This mutant virus, PCMV, differs from myeloproliferative sarcoma virus by two major deletions, one of which spans exactly a 75-base-pair repeat of the long terminal repeat. Functional analysis of recombinant viruses shows that the host-range expansion of PCMV is a property of nucleotide changes within the U3 region of the long terminal repeat. Furthermore, expression assays of chimeric long terminal repeats show that the enhancer region of PCMV joined to the promoter region of Moloney murine leukemia virus is sufficient to direct the synthesis of chloramphenicol acetyltransferase in F9 and PCC4 cells.

Animals↗
PubMed1999

Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K(m) and V(max) values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

Adenosine Triphosphatases↗
PubMed2005

Structure-function analysis of cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats.

The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure-function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative alpha-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave beta-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.

Amino Acid Sequence↗
PubMed2002

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria.

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.

Alkanes↗
PubMed2005

Molecular cloning and functional analysis of a novel 6&1-FEH from wheat (Triticum aestivum L.) preferentially degrading small graminans like bifurcose.

Like barley and other cereals, wheat (Triticum aestivum L.) accumulates branched graminan-type fructans containing both beta-(2,1) and beta-(2,6) fructosyl linkages, mainly with a quite low degree of polymerization (DP). 1&6-kestotetraose (bifurcose) is the major fructan oligosaccharide accumulating in crown tissues and leaves of cereals exposed to chilling. The fructan exohydrolase (FEH) cDNAs 1-FEH w1 and w2 were previously cloned from wheat crowns sampled in mid-November. Here, we report the cloning and functional analysis of another FEH cDNA from a mid-November wheat crown cDNA library. The cDNA encodes a long open reading frame (ORF) of 595 amino acids. Like other FEHs, it has a low iso-electric point (5.2) and it groups together with cell-wall type invertases and not with vacuolar invertases. The deduced amino acid sequence shows 67% identity to wheat 1-FEH w1 and w2. Functional characterization of the recombinant proteins in Pichia pastoris demonstrated that the recombinant enzyme had FEH activity towards the pure compounds 1-kestose, 6-kestose, 1,1-nystose and 1,1,1-kestopentaose. However, when incubated with its putative natural substrates (a mixture of low DP graminans from wheat crowns), it was shown that 1&6-kestotetraose (bifurcose) was preferentially removed from the graminan mixture. High DP wheat graminan and bacterial levan were only poor substrates. No hydrolase activities could be detected towards sucrose and high DP inulin, convincingly demonstrating that the enzyme is not a classic invertase or 1-FEH. The enzyme was termed 6&1-FEH w1. Northern blot analyses showed that 6&1-FEH w1 was expressed in crown tissue from autumn through winter under snow, while the expression levels in leaves were minimal or not detectable. The results strongly suggest that this unique FEH might play an important role in the degradation of branched, low DP wheat graminan (like bifurcose) in wheat crowns in the high fructan content season.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of 9.2 kb left of CRY1 on yeast chromosome III from strain AB972: evidence for a Ty insertion and functional analysis of open reading frame YCR28.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.

Amino Acid Sequence↗
PubMed2006

Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis.

Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution. E6 appears as a chameleon protein combining a conserved structural scaffold with highly variable surfaces participating in generic or specialized HPV functions. We investigated surface residues involved in two specialized activities of high-risk genital HPV E6: p53 tumor suppressor degradation and nucleic acid binding. Screening of E6 surface mutants identified an in vivo p53 degradation-defective mutant that fails to recruit p53 to ubiquitin ligase E6AP and restores high p53 levels in cervical carcinoma cells by competing with endogeneous E6. We also mapped the nucleic acid binding surface of E6, the positive potential of which correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understanding and counteracting the complex pathways of HPV-mediated pathogenesis.

Amino Acid Sequence↗
PubMed1998

Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus.

The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (= IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported.

Azospirillum brasilense↗
PubMed1996

Functional analysis of the preproendothelin-1 gene promoter in pulmonary epithelial cells and monocytes.

At least two human preproendothelin-1 mRNAs are produced by a single gene through the use of different promoters, and the mechanisms controlling the production of these alternative transcripts might be cell-specific. In human lung, endothelin-1 is produced by vascular endothelial cells, bronchial epithelial cells, and pulmonary monocytes/macrophages. Given the important role of endothelin-1 in the pathogenesis of some pulmonary diseases, we carried out a functional analysis of the preproendothelin-1 gene promoter(s) in bronchial epithelial cells and lung monocytes. Two distinct preproendothelin-1 mRNAs were expressed in the two cell populations. Using deletion mutants and enhancer trap transfection experiments, we also identified different regions of the preproendothelin-1 gene necessary for endothelin-1 expression in bronchial epithelial cells and pulmonary monocytes.

Base Sequence↗
PubMed2002

A novel tachykinin-related peptide receptor. Sequence, genomic organization, and functional analysis.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.

Amino Acid Sequence↗
PubMed2004

Identification of a transcriptionally inactive p53 mutant by functional analysis of separated alleles in yeasts (FASAY) in a child osteosarcoma tumor: a case report.

Tumor suppressor gene p53 is one of the most specific genetic alterations occurring in osteosarcoma pathogenesis. It is thought to be an early and key step in the tumorigenesis of osteosarcoma. However, whether the p53 status is a marker predictive of response to therapy and a marker of prognostic value remains controversial. The choice of p53 status detection method certainly account for discrepancies. The authors used a simple functional assay (functional analysis of separated alleles in yeast) on the tumor sample of an 8-year-old girl presenting with an osteosarcoma of the tibia. While making it possible to exclude the presence of a germline mutation, FASAY indicated the presence of a somatic p53 mutation lacking transcriptional activity on p21 and bax target genes. FASAY also strongly suggested a loss of heterozygosity p53, which was confirmed by cytogenetic analysis. Sequencing of cDNA extracted from yeast colonies containing mutated p53 identified a 213 stop mutation in exon 6. Despite these p53 alterations, the child is still in complete remission after a follow-up of 48 months.

Child↗
PubMed2004

Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system.

The ability of Ralstonia solanacearum strain GMI1000 to cause disease on a wide range of host plants (including most Solanaceae and Arabidopsis thaliana) depends on genes activated by the regulatory gene hrpB. HrpB controls the expression of the type III secretion system (TTSS) and pathogenicity effectors transiting through this pathway. In order to establish the complete repertoire of TTSS-dependent effectors belonging to the Hrp regulon and to start their functional analysis, we developed a rapid method for insertional mutagenesis, which was used to monitor the expression of 71 candidate genes and disrupt 56 of them. This analysis yielded a total of 48 novel hrpB-regulated genes. Using the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter fusion system, we provide direct biochemical evidence that five R. solanacearum effector proteins are translocated into plant host cells through the TTSS. Among these novel TTSS effectors, RipA and RipG both belong to multigenic families, RipG defining a novel class of leucine-rich-repeats harbouring proteins. The members of these multigenic families are differentially regulated, being composed of genes expressed in either an hrpB-dependent or an hrpB-independent manner. Pathogenicity assays of the 56 mutant strains on two host plants indicate that, with two exceptions, mutations in individual effectors have no effect on virulence, a probable consequence of genetic and functional redundancy. This large repertoire of HrpB-regulated genes, which comprises > 20 probable TTSS effector genes with no counterparts in other bacterial species, represents an important step towards a full-genome understanding of R. solanacearum virulence.

Bacterial Proteins↗
PubMed2006

Detection and functional analysis of an SNP in the promoter of the human ferritin H gene that modulates the gene expression.

The H ferritin promoter spans approximately 150 bp, upstream of the transcription start and is composed by two cis-elements in position -132 (A box) and -62 (B-box), respectively. The A box is recognized by the transcription factor Sp1, and the B-box by a protein complex called Bbf, which includes the CAAT binding factor NF-Y. In this study we performed a functional analysis of an H ferritin promoter allele carrying a G to T substitution adjacent to the Bbf binding site, in position -69. In vitro studies with reporter constructs revealed a significantly reduced transcriptional activity of this allele compared to that of the w.t. promoter that was mirrored by a decrease in Bbf binding. In vivo, this variant genotype is accompanied by a reduced amount of the H mRNA in peripheral blood lymphocytes.

Alleles↗
PubMed1996

Functional analysis and treatment of cigarette pica.

A series of analyses was conducted to assess and treat the pica of cigarette butts by a young man with mental retardation and autism. First, we demonstrated that pica was maintained in a condition with no social consequences when the available cigarettes contained nicotine but not when the cigarettes contained herbs without nicotine. Second, a choice assessment (Fisher et al., 1992) confirmed that tobacco was preferred over the other components of the cigarette (e.g., paper, filter, etc.). Third, an analogue functional analysis (Iwata, Dorsey, Slifer, Bauman & Richman, 1982/1994) demonstrated that cigarette pica was maintained independent of social consequences. Fourth, a treatment designed to interrupt the hypothesized response-reinforcer relationship reduced consumption of cigarettes to zero. Finally, because cigarette pica occurred primarily when the individual was alone or under minimal supervision, a procedure based on stimulus control was developed to improve the effectiveness of the intervention in these situations.

Adolescent↗
PubMed2004

Structural and functional analysis of the differential effects of c-Jun and v-Jun on prolactin gene expression.

The protooncogene c-Jun and its oncogenic isoform v-Jun are members of the activator protein 1 family of transcription factors that have been shown to have differential transcriptional effects that are both promoter specific and cell type specific. Previously, we have demonstrated that whereas c-Jun inhibits pituitary-specific rat prolactin (rPRL) promoter activity, expression of v-Jun stimulates the rPRL promoter in GH4 pituitary cells. In this report, we have conducted an extensive structure-function analysis of c-Jun vs. v-Jun to determine which regions of these proteins are responsible for their differential transcriptional effects in this pituitary model system. We show that isoform-specific responses are mediated by complex interactions between the delta-domain, serine 243, and the amino-terminal transcriptional activation domains. Thus, in contrast to previous reports, no single domain is responsible for the differential transcriptional activities of c-Jun and v-Jun. Mutation of c-Jun serine 243 to phenylalanine and replacement of the c-Jun amino terminus with the corresponding region from v-Jun, thereby removing the delta-domain, are necessary and sufficient to confer a functional switch from the c-Jun-inhibitory to the v-Jun-activating phenotype. Thus, we propose that isoform-specific subdomains in c-Jun and v-Jun dictate discrete interactions with distinct protein partners, which underlie the differential Jun-dependent transcriptional responses of the rPRL promoter.

Animals↗