Search PubMed⌕ Search

Biomedical subjects

C L Smith

Publications and source records attributed to C L Smith.

At least 19 recordsLinked to original sources
PubMed1997

Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, BcgI.

The BcgI restriction-modification system consists of two subunits, A and B. It is a bifunctional protein complex which can cleave or methylate DNA. The regulation of these competing activities is determined by the DNA substrates and cofactors. BcgI is an active endonuclease and a poor methyltransferase on unmodified DNA substrates. In contrast, BcgI is an active methyltransferase and an inactive endonuclease on hemimethylated DNA substrates. The cleavage and methylation reactions share cofactors. While BcgI requires Mg2+and S -adenosyl methionine (AdoMet) for DNA cleavage, its methylation reaction requires only AdoMet and yet is significantly stimulated by Mg2+. Site-directed mutagenesis was carried out to investigate the relationship between AdoMet binding and BcgI DNA cleavage/methylation activities. Most substitutions of conserved residues forming the AdoMet binding pocket in the A subunit abolished both methylation and cleavage activities, indicating that AdoMet binding is an early common step required for both cleavage and methylation. However, one mutation (Y439A) abolished only the methylation activity, not the DNA cleavage activity. This mutant protein was purified and its methylation, cleavage and AdoMet binding activities were tested in vitro . BcgI-Y439A had no detectable methylation activity, but it retained 40% of the AdoMet binding and DNA cleavage activities.

Base Sequence↗
PubMed1997

Cloning and analysis of the dnaG gene encoding Pseudomonas putida DNA primase.

The dnaG gene coding for primase, a key enzyme in DNA replication, has been isolated from chromosomal DNA of the soil bacterium Pseudomonas putida. It maps within the putative MMS operon, between the rpsU and rpoD genes. Comparison of the deduced amino acid sequence of P. putida DnaG with sequences of other known bacterial primases reveals the presence of a possible regulatory region which would be unique to pseudomonads. The analysis of nucleotide sequence suggests that stable folding of the dnaG mRNA may significantly contribute to the low level of its expression within a cell.

Amino Acid Sequence↗
PubMed1997

Engineering subunit association of multisubunit proteins: a dimeric streptavidin.

A dimeric streptavidin has been designed by molecular modeling using effective binding free energy calculations that decompose the binding free energy into electrostatic, desolvation, and side chain entropy loss terms. A histidine-127 --> aspartic acid (H127D) mutation was sufficient to introduce electrostatic repulsion between subunits that prevents the formation of the natural tetramer. However, the high hydrophobicity of the dimer-dimer interface, which would be exposed to solvent in a dimeric streptavidin, suggests that the resulting molecule would have very low solubility in aqueous media. In agreement with the calculations, a streptavidin containing the H127D mutation formed insoluble aggregates. Thus, the major design goal was to reduce the hydrophobicity of the dimer-dimer interface while maintaining the fundamental structure. Free energy calculations suggested that the hydrophobicity of the dimer-dimer interface could be reduced significantly by deleting a loop from G113 through W120 that should have no apparent contact with biotin in a dimeric molecule. The resulting protein, containing both the H127D mutation and the loop deletion, formed a soluble dimeric streptavidin in the presence of biotin.

Aspartic Acid↗
PubMed1997

Differential activity of progesterone and glucocorticoid receptors on mouse mammary tumor virus templates differing in chromatin structure.

In vivo, transcription factors interact with promoters having complex nucleoprotein structures. The transiently expressed progesterone receptor (PR) efficiently activates a transfected mouse mammary tumor virus (MMTV) promoter but is a poor activator of the MMTV promoter when it acquires an ordered chromatin structure as an endogenous, replicating gene. We show that the deficiency in PR activity is not due to insufficient expression of either B or A isoforms or competition between the two types of MMTV templates. Rather, this deficiency reflects an inability to induce the chromatin remodeling event that is required for activation of the replicated MMTV template. To determine whether this characteristic is common to transiently expressed steroid receptors or specific to the PR, we examined the activity of transiently expressed glucocorticoid (GR) receptor. Unlike the PR, the transiently expressed GR is an effective activator of both MMTV templates and efficiently induces the necessary chromatin remodeling event at the replicated template. These results indicate that the GR and PR have unique requirements for activation of promoters with ordered chromatin structure. These differences may provide a mechanism for establishing target gene specificity in vivo for steroid receptors that recognize and bind to identical DNA sequences.

Animals↗
PubMed1997

Differential display of genome subsets containing specific interspersed repeats.

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.

Base Sequence↗
PubMed1997

Aldosterone and dexamethasone stimulate calcineurin activity through a transcription-independent mechanism involving steroid receptor-associated heat shock proteins.

Heat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.

Adrenalectomy↗
PubMed1997

A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system.

The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal Streptomyces avidinii streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group, D-biotin. The entire construct was designed to allow the soil bacterium Pseudomonas putida to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.

Bacterial Proteins↗
PubMed1997

Sequencing double-stranded DNA by strand displacement.

Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequenced without subcloning, purification or denaturation.

Base Sequence↗
PubMed1997

Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen.

Mixed antiestrogens, such as 4-hydroxytamoxifen (4HT), act as either partial agonists or antagonists of estrogen receptor (ER) function in a tissue-, cell-, and promoter-specific manner, suggesting that intracellular factors modulate their ability to regulate transcription. To determine whether coactivators and corepressors have the capacity to modulate the relative agonist/antagonist activity of 4HT, ER-dependent gene expression was measured in the absence or presence of expression vectors for SRC-1 (steroid receptor coactivator-1) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). In Hep G2 cells in which 4HT is an agonist, exogenous SRC-1 enhanced estradiol (E2)- and 4HT-stimulated transcription in a dose-dependent manner, while SMRT overexpression strongly reduced basal and 4HT-stimulated gene expression with no effect on E2 activity. These observations were not cell- or promoter-specific inasmuch as similar results were obtained in HeLa cells under conditions in which 4HT is an antagonist. A protein-protein interaction assay indicated that the full-length ER binds to SMRT in vitro. To assess whether relative coactivator and corepressor expression within a given cell could modulate the balance of 4HT agonist/antagonist activity, SRC-1 and SMRT were coexpressed. SMRT overexpression blocked SRC-1 coactivation of 4HT-stimulated gene expression and preferentially inhibited 4HT agonist activity whether or not exogenous SRC-1 was present. The cumulative data in this model system indicate that the relative expression of coactivators and corepressors can modulate 4HT regulation of ER transcriptional activity and suggest they could contribute to the tissue-specific ability of mixed antiestrogens to activate or inhibit ER-mediated gene expression.

DNA-Binding Proteins↗
PubMed1996

Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group.

OBJECTIVE: To determine whether a nutritional supplement of selenium will decrease the incidence of cancer. DESIGN: A multicenter, double-blind, randomized, placebo-controlled cancer prevention trial. SETTING: Seven dermatology clinics in the eastern United States. PATIENTS: A total of 1312 patients (mean age, 63 years; range, 18-80 years) with a history of basal cell or squamous cell carcinomas of the skin were randomized from 1983 through 1991. Patients were treated for a mean (SD) of 4.5 (2.8) years and had a total follow-up of 6.4 (2.0) years. INTERVENTIONS: Oral administration of 200 microg of selenium per day or placebo. MAIN OUTCOME MEASURES: The primary end points for the trial were the incidences of basal and squamous cell carcinomas of the skin. The secondary end points, established in 1990, were all-cause mortality and total cancer mortality, total cancer incidence, and the incidences of lung, prostate, and colorectal cancers. RESULTS: After a total follow-up of 8271 person-years, selenium treatment did not significantly affect the incidence of basal cell or squamous cell skin cancer. There were 377 new cases of basal cell skin cancer among patients in the selenium group and 350 cases among the control group (relative risk [RR], 1.10; 95% confidence interval [CI], 0.95-1.28), and 218 new squamous cell skin cancers in the selenium group and 190 cases among the controls (RR, 1.14; 95% CI, 0.93-1.39). Analysis of secondary end points revealed that, compared with controls, patients treated with selenium had a nonsignificant reduction in all-cause mortality (108 deaths in the selenium group and 129 deaths in the control group [RR; 0.83; 95% CI, 0.63-1.08]) and significant reductions in total cancer mortality (29 deaths in the selenium treatment group and 57 deaths in controls [RR, 0.50; 95% CI, 0.31-0.80]), total cancer incidence (77 cancers in the selenium group and 119 in controls [RR, 0.63; 95% CI, 0.47-0.85]), and incidences of lung, colorectal, and prostate cancers. Primarily because of the apparent reductions in total cancer mortality and total cancer incidence in the selenium group, the blinded phase of the trial was stopped early. No cases of selenium toxicity occurred. CONCLUSIONS: Selenium treatment did not protect against development of basal or squamous cell carcinomas of the skin. However, results from secondary end-point analyses support the hypothesis that supplemental selenium may reduce the incidence of, and mortality from, carcinomas of several sites. These effects of selenium require confirmation in an independent trial of appropriate design before new public health recommendations regarding selenium supplementation can be made

Adult↗
PubMed1996

CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription.

Steroid receptors are ligand-regulated transcription factors that require coactivators for efficient activation of target gene expression. The binding protein of cAMP response element binding protein (CBP) appears to be a promiscuous coactivator for an increasing number of transcription factors and the ability of CBP to modulate estrogen receptor (ER)- and progesterone receptor (PR)-dependent transcription was therefore examined. Ectopic expression of CBP or the related coactivator, p300, enhanced ER transcriptional activity by up to 10-fold in a receptor- and DNA-dependent manner. Consistent with this, the 12S E1A adenoviral protein, which binds to and inactivates CBP, inhibited ER transcriptional activity, and exogenous CBP was able to partially overcome this effect. Furthermore, CBP was able to partially reverse the ability of active ER to squelch PR-dependent transcription, indicating that CBP is a common coactivator for both receptors and that CBP is limiting within these cells. To date, the only other coactivator able to significantly stimulate receptor-dependent transcription is steroid receptor coactivator-1 (SRC-1). Coexpression of CBP and SRC-1 stimulated ER and PR transcriptional activity in a synergistic manner and indicated that these two coactivators are not functional homologues. Taken together, these data suggest that both CBP and SRC-1 may function in a common pathway to efficiently activate target gene expression.

CREB-Binding Protein↗
PubMed1996

Dopaminergic regulation of progesterone receptors: brain D5 dopamine receptors mediate induction of lordosis by D1-like agonists in rats.

To characterize the signaling pathway by which the neurotransmitter dopamine modulates progesterone receptor (PR) activation, the steroid-dependent behavior lordosis was used in estrogen-primed ovariectomized Sprague-Dawley rats with stereotaxic implanted third ventricle cannulas. Lordosis was observed in response to solicitous males in females after central administration of the D1-like agonist SKF38393 and three of its analogs (SKF77434, SKF75640, and SKF85174). In contrast, D1-like antagonist SCH23390 and D1-like/D2 repopulation inhibitor EEDQ blocked behavior inducible by the D1-like agonists. Further, antisense oligonucleotides to D5, but not D1, dopamine receptor mRNA suppressed reproductive behavior associated with D1-like stimulation. This finding provides strong evidence that dopaminergic modulation of lordosis is mediated by the novel D5 dopamine receptor. Although D1, but not D5, dopamine receptor mRNAs were detected in the ventromedial nucleus (VMN) by in situ hybridization, agonists microinjected into the VMN, but not into the arcuate nucleus or preoptic area, induced lordosis, suggesting the functional presence of D5 dopamine receptors in the VMN. Also in support, D5 receptor mRNA antisense microinjected into the VMN blocked the subsequent induction of lordosis by D1-like agonists. Finally, facilitation of sex behavior by D1-like agonists was blocked by the antiprogestin RU38486 and PR antisense oligonucleotide. Collectively, the data provide strong evidence for dopaminergic modulation of reproductive behavior through D5 dopamine receptor-mediated modulation of PR-dependent behavior in rat CNS.

Animals↗
PubMed1996

Diffusional mobility of Golgi proteins in membranes of living cells.

The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged from 3 x 10(-9) square centimeters per second to 5 x 10(-9) square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes, which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.

Aluminum Compounds↗
PubMed1996

The development of microfabricated arrays for DNA sequencing and analysis.

Microfabricated arrays of immobilized oligodeoxynucleotide probes are proving to be a powerful tool for rapidly generating sequence data via hybridization. These arrays are made either by immobilization of the probe post-synthetically, or by in situ synthesis of the probe. Hybridization of the target is easily achieved on the arrays, with analysis proceeding either by direct detection, or through enzyme-mediated detection; analysis of the hybridization pattern yields sequence information about the target. Such facile and rapid data acquisition will assist the challenging task of sequencing the human genome, and also will lead to a new generation of diagnostic assays.

Biotechnology↗
PubMed1996

An analysis of the elements essential in the development of a customized TBI program.

Bone marrow transplant, once a procedure carried out at a relatively small number of large medical centers, is being employed at an increasing number of non-academic hospitals. The use of total body photon irradiation for the purpose of marrow ablation and immunosuppression can be expected to accompany the outward growth of bone marrow transplantation at these same hospitals. An analysis of the fundamental physical factors associated with total body photon irradiation (TBI) should be of value to facilities considering the incorporation of a total body irradiation program into an existing radiation oncology department. By examining the existing resources of a radiotherapy center and the clinical objectives of the treatment, a facility considering TBI may determine whether or not their center already fulfills the necessary criteria, in addition to the avoidance of potential pitfalls in project implementation.

Bone Marrow Purging↗
PubMed1996

Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

Animals↗