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Biomedical subjects

C F Wright

Publications and source records attributed to C F Wright.

At least 19 recordsLinked to original sources
PubMed2006

Members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt.

In an adenosine triphosphate (ATP)-dependent process, the hSWI/SNF chromatin remodeling complex functions to alter chromatin structure, thereby regulating transcription factor access to DNA. In addition to interactions with transcription factors and recognition of acetylated histone residues, the chromatin remodeling activity of hSWI/SNF has also been shown to respond to a variety of cell signaling pathways. Our results demonstrate a novel interaction between the serine/threonine kinase Akt and members of the hSWI/SNF chromatin remodeling complex. Activation of Akt in HeLa cells resulted in its association with hSWI/SNF subunits: INI1, BAF155 and BAF170, as well as actin. BAF155 became preferentially recognized by an antibody that detects phosphorylated Akt substrates upon activation of Akt, suggesting that BAF155 may be an in vivo target for phosphorylation by Akt. Glutathione-S-transferase (GST) pulldown experiments demonstrated that INI1 and BAF155 were both capable of directly interacting with Akt. Finally, in vitro kinase assays provided additional evidence that BAF155 and potentially INI1 are substrates for Akt phosphorylation. These data provide the first evidence that Akt signaling may modulate function of the hSWI/SNF complex.

Actins↗
PubMed2005

Heat- and lignosulfonate-treated canola meal as a source of ruminal undegradable protein for lactating dairy cows.

This experiment used 18 lactating Holstein cows in a 3 x 3 Latin square replicated 6 times to determine the effectiveness of processing with moist heat or moist heat combined with lignosulfonate (LSO3) for increasing the ruminal undegradable fraction of canola meal for use as a protein supplement for lactating dairy cows. Diets were formulated to be isonitrogenous and contained one of 3 forms of canola meal; untreated canola meal (UCM), heat-treated canola meal (HTCM) or heat-and LSO3-treated canola meal (LSO3CM). Total collection of urine and feces was taken from each cow during the last 5 d of each 42-d experimental period. Milk production was greater for cows fed the LSO3CM diet (36.6 kg/d) than for cows fed the UCM diet (34.8 kg/d) but did not differ from cows fed the HTCM diet (35.3 kg/d). Digestibility of crude protein was lower for cows supplemented with LSO3CM and they had reduced concentrations of ruminal ammonia N, blood urea N, and milk urea N compared with cows supplemented with UCM or HTCM. Dry matter intake and apparent digestibilities of neutral and acid detergent fiber were increased in cows fed the LSO3CM diet. Urinary N excretion (as % of N intake) was reduced in cows fed the LSO3CM diet. These results indicate that moist heat combined with LSO3 treatment of canola meal was effective in increasing the proportion of crude protein digested in the lower digestive tract of lactating cows and was therefore used more effectively as a source of protein than UCM or HTCM.

Ammonia↗
PubMed2001

Vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins.

Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late. Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter. VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity. Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation. Overexpression and purification of these proteins from Escherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions. These studies identify two host cell factors potentially contributing to poxvirus replication in vivo.

Amino Acid Sequence↗
PubMed1998

A vaccinia virus late transcription factor with biochemical and molecular identity to a human cellular protein.

A factor designated VLTF-X is required to support vaccinia virus late transcription in vitro. It has been found that a late promoter DNA binding activity cochromatographs and cosediments with VLTF-X activity. Current experiments show that VLTF-X activity is present in a variety of uninfected mammalian cell types and is indistinguishable from that recovered from infected cells based upon several criteria. VLTF-X activity from both sources displays the same purification profile over phosphocellulose and DNA affinity resins and has the same sedimentation coefficient. In addition, the factors purified from both infected and uninfected cells form protein-DNA complexes of identical electrophoretic mobility in the presence of vaccinia virus late promoter-containing DNA. The affinity of these factors for the late promoter probes is identical and late promoter-specific based on competition experiments. Moreover, VLTF-X purified from both sources bound to late promoter-containing DNA in the presence or absence of MgCl2 and ATP and formed complexes resistant to heat inactivation. These experiments offer proof that vaccinia virus factor VLTF-X is a host cell protein that supports transcription of the viral late genes.

Adenosine Triphosphate↗
PubMed1998

A vaccinia virus late transcription factor copurifies with a factor that binds to a viral late promoter and is complemented by extracts from uninfected HeLa cells.

We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.

HeLa Cells↗
PubMed1998

Establishment and molecular characterization of five cell lines derived from renal and extrarenal malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy. To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presenting with MRT (two renal and three extrarenal tumors). All of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. All are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss of heterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated. Although mutations of the p53 gene were not detectable by sequence analysis, all of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype of these tumors.

Adolescent↗
PubMed1996

Discovery of the hemifumarate and (alpha-L-alanyloxy)methyl ether as prodrugs of an antirheumatic oxindole: prodrugs for the enolic OH group.

Ether, ester, and carbonate derivatives of the antirheumatic oxindole 1 were prepared and screened as potential prodrugs of 1. This effort led to the discovery of the (alpha-L-alanyloxy)-methyl ether and hemifumarate derivatives of 1 which deliver the drug efficiently into the circulation of test animals, are stable in the solid state, and possess good stability in solution at low pH as required to ensure gastric stability. Success in achieving acceptable bioavailabilities of 1 across species (rats, dogs, and monkeys) followed the inclusion of ionizable functionality within the promoiety to compensate for masking the polar enolic OH group of the free drug. However, the introduction of ionizable functionality was often associated with decreased stability, as demonstrated by the hemisuccinate, hemiadipate, hemisuberate, and alpha-amino ester derivatives of 1 which could not be isolated. A clear exception was the hemifumarate derivative of 1 which was not only isolable but actually more stable at neutral pH than the nonionizable ester analogues. The solution and solid state stability of the hemifumarate, together with its activity as a prodrug of 1, suggests that hemifumarate be considered as an alternative to hemisuccinate as a prodrug derivative for alcohols, particularly in situations where solution state stability is an issue.

Animals↗
PubMed1996

MDM2 amplification, P53 mutation, and accumulation of the P53 gene product in malignant fibrous histiocytoma.

Fifty-two cases of malignant fibrous histiocytoma (MFH) were evaluated for amplification of the MDM2 gene, mutation of the P53 gene, accumulation of the P53 gene product, and their relation to disease-free and overall survival. All tests were carried out on formalin-fixed, paraffin-embedded tissue samples. Amplification of the MDM2 gene was detected in 15 of 52 cases (29%). Six of 52 cases (12%) demonstrated abnormalities of the P53 gene. Sequence analysis detected point mutations in four cases and a 1-base pair deletion in one case, whereas differential polymerase chain reaction (dPCR) indicated that the P53 gene had been entirely deleted in one case. Eight of 52 cases (15%) demonstrated staining for the P53 protein in >10% of tumor cells. The presence of MDM2 amplification did not have a significant effect on either disease-free or overall survival. Patients with accumulation of the P53 gene product did not differ in disease-free or overall survival from patients without P53 accumulation. Survival also was not significantly different in patients with genetic aberration in P53. However, when the patients were stratified by histologic grade, the results indicated that patients with alterations in the P53 gene may have shorter overall survival.

Gene Amplification↗
PubMed1996

The A2L intermediate gene product is required for in vitro transcription from a vaccinia virus late promoter.

Previously, the in vitro late transcription system of vaccinia virus was resolved into four components: the 17- and 30-kDa products of the A1L and G8R intermediate genes, respectively, the viral DNA-dependent RNA polymerase, and an unmapped factor sedimenting at 32 to 38 kDa. Another protein, the 26-kDa product of the A2L open reading frame was predicted to be a late transcription factor on the basis of a transient-expression assay but was not recognized as being necessary for transcriptional activity in vitro. We now report that both the unmapped factor and the 26-kDa protein are required for transcription from a vaccinia virus late promoter in vitro. Since the 26-kDa protein has now been shown to be a trans-activator of late transcription and it is the product of a known gene, we suggest that it be designated VLTF-3.

Animals↗
PubMed1995

Use of semiquantitative PCR to assess onset and treatment of Pneumocystis carinii infection in rat model.

The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.

Animals↗
PubMed1993

Purification of the late transcription system of vaccinia virus: identification of a novel transcription factor.

We have resolved the in vitro late transcription system of vaccinia virus into four components consisting of RNA polymerase and three accessory factors. One of these additional factors is a 30-kDa protein which was previously shown to be required for late transcription in vitro and was indirectly shown to be the product of the G8R open reading frame. Another factor, of 17 kDa, was previously identified as a possible late transcription factor by an assay which demonstrated that the gene encoding it, A1L, was required for late gene expression in vivo. The G8R and A1L open reading frames have now been cloned into a baculovirus expression system, and the corresponding proteins have been purified. Both are necessary for late transcription in vitro, confirming that these intermediate genes encode late transcription factors. The third factor has a sedimentation coefficient consistent with a protein of 32 to 38 kDa. Experimental results suggest that this is a previously unidentified factor encoded by a vaccinia virus early gene. The RNA polymerase functioning in this system was purified from vaccinia virus-infected cells; however, it can be complemented by the RNA polymerase which is packaged in virions. The three smaller proteins and RNA polymerase are all necessary, and together are sufficient, for the synthesis of late viral mRNA in vitro.

Base Sequence↗
PubMed1991

A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene.

A factor, designated VLTF-1, that is required in vitro for specific transcription of vaccinia virus late genes was previously isolated from vaccinia virus-infected cells. Subsequent genetic experiments identified three vaccinia virus genes, encoding proteins of 17, 26, and 30 kDa, that together trans activate late gene expression in vivo. The purpose of this study was to determine whether VLTF-1 corresponded to one of the three trans activators. Toward this end, VLTF-1 was further purified, the trans-activator genes were expressed in Escherichia coli, and antisera were made to the native and recombinant proteins. Antibody to the 30-kDa recombinant protein reacted on Western immunoblots with a protein of approximately Mr 30,000 that cochromatographed and cosedimented with VLTF-1 activity from virus-infected cells. Conversely, antibody to purified VLTF-1 bound to products produced by in vitro transcription and translation of the open reading frame encoding the 30-kDa trans-activator protein. Both antisera depleted VLTF-1 activity and blocked late gene transcription by partially purified extracts of vaccinia virus-infected cells. Taken together, these data demonstrate that the 30-kDa trans activator comprises part, if not all, of VLTF-1 activity.

Blotting, Western↗
PubMed1991

Detection of Epstein-Barr virus sequences in Hodgkin's disease by the polymerase chain reaction.

The authors examined paraffin-embedded lymph node biopsies from 65 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, using the highly sensitive polymerase chain reaction technique. Overall 40% of the cases were positive for EBV DNA; there were no statistically significant differences in the frequency of EBV positivity among the different subtypes of Hodgkin's disease. These results are in agreement with those of previous studies that employed less sensitive detection techniques and suggest that EBV either is present in pathologic tissues only in some phases of the evolution of Hodgkin's disease or is a pathogenetic factor involved in only a portion of cases.

Biopsy↗
PubMed1989

Identification of factors specific for transcription of the late class of vaccinia virus genes.

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.

Adenosine Triphosphate↗
PubMed1988

Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants.

Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.

Amino Acids↗
PubMed1988

Autoregulation of the yeast copper metallothionein gene depends on metal binding.

The yeast CUP1 gene product, copper metallothionein, acts to repress the basal transcription of its own structural gene. By creating a series of truncation and amino acid substitutions in CUP1, we show that the ability of the protein to autoregulate is directly correlated to its ability to bind and detoxify copper. These results support a model in which metallothionein controls the level of free intracellular copper available to interact with positive transcription factors. In addition, mutations in chemically equivalent cysteine residues were functionally dissimilar, suggesting that partial sites in the molecule are critical for the formation of the sulfur-metal cluster.

Carrier Proteins↗