PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Protein Array Analysis”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMedDate not supplied

Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.

Genetic analysis of eukaryotic transcriptional promoters has revealed that protein-coding genes often contain a complex array of cis-control elements consisting of upstream activator sequences and enhancer elements. The metallothionein genes provide a useful example for dissecting the action of multiple interspersed control elements that govern both basal level and regulated expression in animal cells. The human metallothionein (hMTIIA) promoter has been analysed in detail and found to contain no less than five distinct control elements in the 5' flanking regions of the gene that mediate specificity and regulation of transcription. These different control elements can be functionally subdivided into two categories: basal and induced elements. There are several distinct basal recognition sequences, which include a TATA-box, a GC-box, and at least two basal level enhancer (BLE) sequences, that function like classical enhancer elements. The hMTIIA gene also responds to induction by heavy metals and by steroid hormones through the action of metal regulatory elements (MRE) and glucocorticoid responsive elements (GRE). Here we report the identification of two cellular DNA-binding proteins that interact selectively with sequences governing the basal level expression of hMTIIA. One of these factors is a novel activator protein (AP1) that interacts with sequences in the BLE of hMTIIA and also binds to a site within the 72-base pair (bp) repeats of the simian virus 40 (SV40) enhancer region. The second protein has been purified to homogeneity and shown to be transcription factor Sp1 which recognizes and binds to a single GC-box element within the hMTIIA promoter.

Base Sequence↗
PubMed2026

Exploring the therapeutic targets and signaling mechanisms of quercetin activity against radiation skin ulcer based on the observational research of network pharmacology.

Radiation skin ulcer is a common adverse complication after radiotherapy. Currently, there is no efficient therapy for this complication. In this study, we searched for the potential pathological targets of radiation skin ulcer and the potential pharmacological targets of quercetin, respectively, and obtained the potential therapeutic targets after intersection. Subsequently, an array of bioinformatics assessments on possible therapeutic targets was conducted, encompassing functional enrichment studies, analysis of protein interaction networks, identification of key targets, and validation through molecular docking. The enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways shows that the therapeutic effect of quercetin on radiation skin ulcer may be through targeting aging cells. In addition, we identified 5 core targets, including AKT1, EGFR, MAPK3, SRC, and TP53. They are significantly enriched in EGFR tyrosine kinase inhibitors (SRC, AKT1, EGFR, and MAPK3) and epidermal growth factor receptor signaling pathways (SRC, EGFR, and AKT1), indicating the importance of EGFR signaling. Quercetin may have a therapeutic effect on radiation skin ulcer by targeting aging cells. Specifically, it may act through 4 core targets, including AKT1, EGFR, SRC, and TP53.

Quercetin↗
PubMed1988

Transcriptional analysis of the major surface array gene of Caulobacter crescentus.

The major component of the paracrystalline surface array of Caulobacter crescentus CB15 and one of the most abundant cellular proteins is a protein designated 130K. We have determined the DNA sequence of the 5' portion of the 130K gene, including the N-terminal one-third of the protein coding region, and analyzed the transcription of the gene. The site of transcription initiation was determined by S1 mapping of Caulobacter RNA. Although the DNA sequence upstream from the transcription start site showed significant homology to the consensus promoter sequences of Escherichia coli, S1 analysis of RNA from E. coli carrying the 130K gene on a plasmid indicated that the 130K promoter was not transcribed by E. coli RNA polymerase in vivo. Quantitative S1 analysis of RNA isolated from synchronously growing Caulobacter cells suggested that this promoter was not under developmental regulation; the amount of 130K transcript varied no more than 1.5-fold during the cell cycle. The length of the 130K mRNA was determined to be 3.3 kilobases by Northern (RNA blot) analysis, indicating that the 130K mRNA is not part of a polycistron. The amino acid sequence predicted from the DNA sequence agreed well with the N-terminal amino acid sequence determined by sequencing of the 130K protein. The 130K protein appears to be synthesized without an N-terminal leader sequence, but the N-terminal 20 amino acids are relatively hydrophobic and may function like a signal sequence during transmembrane translocation.

Amino Acid Sequence↗
PubMed1987

Expression of normal and mutant ras proteins in human acute leukemia.

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.

Acute Disease↗
PubMed1997

Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein.

We report the expression and purification of recombinant rodent malarial Plasmodium yoelii circumsporozoite surface protein (PyCSP) in Escherichia coli. To facilitate purification of the recombinant protein, the PyCSP was expressed as an amino-terminal fusion protein to glutathione S-transferase and as a carboxy-terminal fusion protein to a hexahistidyl tag. The expression of the fusion protein was controlled by the inducible tac promoter. Under optimal conditions the immunoreactive PyCSP represented approximately 0.04% of the total cell lysate. Western blot analysis probing with an anti-PyCSP antibody revealed a wide array of immunoreactive bands. Material isolated by affinity purification on glutathione-Sepharose 4B resin also contained multiple bands indicative of premature termination or carboxyl-terminal degradation. Analysis of protein retained on a nickel nitrilotriacetic acid resin revealed evidence of amino-terminal deleted material. Combining the two mild affinity purifications resulted in isolation of a single immunoreactive protein of approximate molecular weight of 96 kDa. We anticipate that the approach described in this study will facilitate the production of highly purified recombinant proteins.

Animals↗
PubMed1998

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.

Journal Article↗
PubMed1993

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.

Bacterial Proteins↗
PubMed1991

2D crystal forms of annexin IV on lipid monolayers.

Two-dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 x 18 nm and 28 x 28 nm. The former unit cell was similar to a previously described form of annexin VI. The implications of these observations are discussed.

Annexins↗
PubMed1995

Identification of the sequences in HMG-CoA reductase required for karmellae assembly.

In all eukaryotic cells that have been examined, specific membrane arrays are induced in response to increased levels of the ER membrane protein, HMG-CoA reductase. Analysis of these inducible membranes has the potential to reveal basic insights into general membrane assembly. Yeast express two HMG-CoA reductase isozymes, and each isozyme induces a morphologically distinct proliferation of the endoplasmic reticulum. The isozyme encoded by HMG1 induces karmellae, which are long stacks of membranes that partially enclose the nucleus. In contrast, the isozyme encoded by HMG2 induces short stacks of membrane that may be associated with the nucleus, but are frequently present at the cell periphery. To understand the molecular nature of the different cellular responses to Hmg1p and Hmg2p, we mapped the region of Hmg1p that is needed for karmellae assembly. For this analysis, a series of exchange alleles was examined in which a portion of the Hmg2p membrane domain was replaced with the corresponding Hmg1p sequences. Results of this analysis indicated that the ER lumenal loop between predicted transmembrane domains 6 and 7 was both necessary and sufficient for karmellae assembly, when present in the context of an HMG-CoA reductase membrane domain. Immunoblotting experiments ruled out the simple possibility that differences in the amounts of the various chimeric HMG-CoA reductase proteins was responsible for the altered cellular responses. Our results are consistent with the hypothesis that each yeast isozyme induces or organizes a qualitatively different organization of ER membrane.

Amino Acid Sequence↗
PubMed1992

Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain.

The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.

Aeromonas hydrophila↗
PubMed1982

The covalent structure of apolipoprotein A-I from canine high density lipoproteins.

The complete amino acid sequence of apolipoprotein A-I (apo-A-I) from canine serum high density lipoproteins (HLD) has been determined by automated Edman degradation of the intact protein and proteolytic fragments derived therefrom. The major strategy involved analysis of overlapping sets of peptides generated by cleavage at lysyl residues with Myxobacter protease and by tryptic hydrolysis at arginines in the citraconylated protein derivative. Canine apo-A-I has 232 residues in its single polypeptide chain and its covalent structure is highly homologous to one of the two reported sequences for human apo-A-I. As in the case for the human apoprotein, predictive analysis of the canine apo-A-I sequence suggests that it comprises a series of amphiphilic alpha helices punctuated by a periodic array of prolyl residues. Human HDL contains a second major protein component, apolipoprotein A-II (apo-A-II) that is lacking in HDL from dog serum. The absence of apo-A-II in canine HDL raised the possibility that the apo-A-I from this source might contain within its primary structure sequences related to apo-A-II and thus perform the dual function of both proteins in one. Our analysis proves that canine apo-A-I has all of the structural features of human apo-A-I and that it is not an A-I: A-II hybrid molecule.

Amino Acid Sequence↗
PubMed1990

Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography.

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit.

1-Propanol↗
PubMed1998

Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.

Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.

Amino Acid Sequence↗
PubMed1994

Oligonucleotide arrays: new concepts and possibilities.

Advances in solid-phase oligonucleotide synthesis and hybridization techniques have led to an incipient technology based on the use of oligonucleotide arrays. The inclusion of a large number of oligonucleotide probes within a single array greatly reduces the cost of their synthesis and allows thousands of hybridizations to be carried out simultaneously. The range of potential applications of oligonucleotide arrays was expanded by the realization that nucleic acids can be sequenced by hybridizing them to all possible oligonucleotides of a given length. Additional possibilities are offered by novel types of oligonucleotide arrays that are capable of parallel sorting, isolating, and manipulating thousands, and even millions, of nucleic acid species. Fields, such as site-directed mutagenesis, protein engineering, and recombinant DNA technology, would benefit from using these arrays. Further, these approaches could enable the analysis of entire genomes by preparing ordered fragment libraries, and by sequencing complex pools of nucleic acids, in a novel approach that provides long-range sequence information by generating nested nucleic acids and then surveying the oligonucleotides contained in the nested strands. This would allow large diploid genomes to be sequenced directly in a completely automated procedure that does not require fragment cloning or chromosome mapping.

Alleles↗
PubMed1998

Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map.

Identification of all human protein-protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein-protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein-protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.

Chromosome Mapping↗