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Biomedical subjects

C L Smith

Publications and source records attributed to C L Smith.

At least 73 records · Page 4Linked to original sources
PubMed1999

Clinical usefulness of 3% hydrogen peroxide in acute upper GI bleeding: a pilot study.

BACKGROUND: A major problem in the endoscopic management of acute upper gastrointestinal (GI) bleeding is the presence of blood and clots overlying the bleeding source, preventing visualization of the lesion. A simple alternative is to alter the characteristics of blood such that it not only becomes easier to remove but also becomes translucent. We report the results of a pilot study on the use of hydrogen peroxide in patients with acute upper GI bleeding. METHODS: Patients with acute upper GI bleeding were studied if the presence of blood or clots obscured the site of bleeding. The potential site of bleeding was initially sprayed with 200 mL water and then with 200 mL 3% hydrogen peroxide mixed with simethicone. RESULTS: In 6 patients with acute upper GI bleeding, hydrogen peroxide spray resulted in good to excellent visualization of the bleeding source. Hemostasis occurred in 2 patients who were actively bleeding. There were no adverse effects or complications. CONCLUSIONS: Hydrogen peroxide significantly enhanced clot dissolution and endoscopic visualization in patients with acute upper GI bleeding.

Acute Disease↗
PubMed1999

Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection.

A new method of amplifying short DNA molecules immobilized on a solid support has been developed. This method uses a solid-phase rolling circle replication reaction, termed rolling circle amplification (RCA). The probe consists of a single-stranded DNA primer anchored at the 5' terminus to a solid support and a single stranded DNA template hybridized to the immobilized primer. Here, DNA ligase was used to circularize the template, and DNA polymerase I was used to extend the immobilized primer in a rolling circle replication reaction. This method was used to identify a known polymorphism in BRCA1 exon 5. These results demonstrate that RCA offers considerable promise to facilitate effective mutation screening of DNA using a solid-phase format.

DNA↗
PubMed1999

Differential sequencing with mass spectrometry.

Differential or genetic sequencing requires searching sample DNA for variations with respect to a reference sequence. Conventional detection techniques are too labor and cost expensive for use in diagnostic applications, therefore new technologies will be required. Measurement techniques based on mass spectrometry (MS) possess the potential for high-throughput, high fidelity measurement of sequence variation. Unambiguous detection of polymorphic sequences has been demonstrated, even in heterozygous samples. Automated reproducible measurements of microscopic arrays of samples will enable the high-throughput detection required for large-scale applications. Computational simulation and analysis of experimental parameters prior to experimentation will provide the optimization necessary for development of robust, reproducible measurements.

Mass Spectrometry↗
PubMed1999

Discrimination of DNA duplexes with matched and mismatched tandem repeats by T4 endonuclease VII.

A simple in situ method for scoring short tandem DNA repeat length has been developed using T4 endonuclease VII. This method measures tandem repeated simple sequences embedded in unique sequences. Single-stranded loops are formed on duplexes containing mismatched (different) numbers of tandem repeats. No single stranded loops are formed on structures containing matched (identical) numbers of tandem repeats. The matched and mismatched loop structures were distinguished and differentially labeled by enzymatic treatment with T4 endonuclease VII.

Bacteriophage T4↗
PubMed1999

PCR based targeted genomic and cDNA differential display.

We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.

Alleles↗
PubMed1999

Biopharmaceutical approaches for developing and assessing oral peptide delivery strategies and systems: in vitro permeability and in vivo oral absorption of salmon calcitonin (sCT).

PURPOSE: To evaluate a biopharmaceutical approach for selecting formulation additives and establishing the performance specifications of an oral peptide delivery system using sCT as a model peptide. METHODS: The effect of formulation additives on sCT effective permeability and transepithelial electrical resistance (TEER) was evaluated in side-by-side diffusion chambers using rat intestinal segments. Baseline regional oral absorption of sCT was evaluated in an Intestinal and Vascular Access Port (IVAP) dog model by administration directly into the duodenum, ileum, and colon by means of surgically implanted, chronic catheters. The effect of varying the input rate and volume of the administered solution on the extent of sCT absorption was also evaluated. Citric acid (CA) was utilized in all studies to cause a transient reduction in local pH. In vitro samples and plasma samples were analyzed by radioimmunoassay (RIA). Two oral delivery systems were prepared based on the results of the in vitro and IVAP studies, and evaluated in normal dogs. RESULTS: Maximal permeability enhancement of sCT was observed using taurodeoxycholate (TDC) or lauroyl carnitine (LC) in vitro. Ileal absorption of sCT was higher than in other regions of the intestine. Low volume and bolus input of solution formulations was selected as the optimal condition for the IVAP studies since larger volumes or slower input rates resulted in significantly lower sCT bioavailability (BA). Much lower BA of sCT was observed when CA was not used in the formulation. The absolute oral bioavailability (mean+/-SD) in dogs for the control (sCT + CA) and two proprietary sCT delivery systems was 0.30%+/-0.05%, 1.10+/-0.18%, and 1.31+/-0.56%, respectively. CONCLUSIONS: These studies demonstrate the utility of in vitro evaluation and controlled in vivo studies for developing oral peptide delivery strategies. Formulation additives were selected, the optimal intestinal region for delivery identified, and the optimal release kinetics of additives and actives from the delivery system were characterized. These methods were successfully used for devising delivery strategies and fabricating and evaluating oral sCT delivery systems in animals. Based on these studies, sCT delivery systems have been fabricated and tested in humans with favorable results.

Administration, Oral↗
PubMed1999

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product.

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

Amino Acid Sequence↗
PubMed1999

A ligand binding domain mutation in the mouse glucocorticoid receptor functionally links chromatin remodeling and transcription initiation.

We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

Amino Acid Sequence↗
PubMed1999

The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily.

In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitin-protein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway.

Angelman Syndrome↗
PubMed1999

Differential localization and activity of the A- and B-forms of the human progesterone receptor using green fluorescent protein chimeras.

Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.

Animals↗
PubMed1998

Functional subnuclear partitioning of transcription factors.

After many years of reductionistic approaches to characterize molecular mechanisms involved in transcription, the number of factors recognized to take part in this process has increased remarkably and continues to grow. When considering posttranslational modifications in conjunction with the large number of factors involved in modulating the activity of transcription complex components, the overall intricacy becomes staggering. After two decades of intensive molecular investigations, there has been a concerted effort to integrate these findings with cellular approaches to understand transcription on a more global level. This sort of reasoning actually revisits studies of approximately 20 years ago that considered the functional consequences of steroid receptor association with nuclear structure. With an abundance of new molecular probes and increasingly powerful instruments to detect them in fixed and, more recently, live cells, the issue of functional subnuclear organization is receiving increased attention. In this report, we focus on advances in characterizing the functional significance of transcription factor association with the nucleoskeleton. In particular, we consider recent biochemical and "molecular morphology" data that point to the importance of dynamic spatial and solubility partitioning of gene regulators with nuclear architecture.

Cell Nucleus↗
PubMed1998

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR.

Base Sequence↗
PubMed1998

Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites.

We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.

Animals↗
PubMed1998

Breast cancer mortality trends in Australia: 1921 to 1994.

OBJECTIVE: To analyse breast cancer mortality trends in Australia and to see if mammographic screening has yet led to a reduction in mortality. DESIGN: Retrospective analysis of trends in mortality rates from breast cancer in Australian women between 1921 and 1994, and in potentially explanatory variables such as fertility, body size, age at menarche, and screening. RESULTS: Changes in breast cancer mortality in Australian women could not be explained by chance variation alone. Mortality rose steadily (average annual increase, 1.0%) to 1940-1944, fell to the 1960s and early 1970s, and rose (average annual increase, 0.3%) to the late 1980s. Between 1985-1989 and 1990-1994, breast cancer mortality fell by 3.2% in women 50-69 years of age (the target age group for mammographic screening) and by 4.2% in women 25-49 years of age. There was almost on change (-0.2%) in breast cancer mortality in older women in this period. The proportion of women screened in all age groups increased substantially between 1988 and 1994; nearly 65% of women in the target age group had had at least one mammogram by 1994. Decreases in fertility were followed by increases in mortality, and vice versa. CONCLUSIONS: Trends in breast cancer mortality have probably been influenced by changing fertility, nutrition and body-size increases among Australian women. Improvements in stage at diagnosis and treatment have probably moderated the upwards pressure on mortality caused by an increasing incidence. Recent falls in mortality could be expected to continue as more women participate in the mammographic screening program. This trend should be more clearly evident in the second half of the 1990s.

Adult↗
PubMed1998

Advances in DNA diagnostics.

The key advances in DNA diagnostics during the past year are techniques which will lead to advanced throughput without sacrificing sensitivity: miniaturization of samples to reduce material cost and preparation time, and parallelization through use of measurement arrays. The most promising gains have come in the areas of DNA arrays and mass spectrometry, where differential sequencing measurements are now possible.

Diagnostic Techniques and Procedures↗
PubMed1998

A review of digital image networking technologies for radiation oncology treatment planning.

Dosimetrists, physicists, and physicians rely heavily upon digital imaging modalities in their daily work. In the current healthcare marketplace, many radiotherapy facilities operate satellite centers or may be undergoing mergers with other service providers in their community. As a consequence, the development of network-based radiotherapy image communication may yield a significant impact on the clinical operation of such centers. Digital image networking will become an everyday tool in radiotherapy treatment planning in the near future. As responsible users of this technology, an accurate perception of what the network does is essential and enhances our ability to utilize it. This article presents a review of the network architecture and transmission standards necessary for understanding and developing a radiotherapy image network.

Computer Communication Networks↗
PubMed1998

Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily.

Upon binding to regulatory elements in mammalian chromosomes, steroid receptors induce specific transitions in the nucleoprotein structure of the template. These transitions reflect, in part, the reorganization of chromatin structure to permit interaction of secondary factors with target sequences in promoter regulatory regions. Steroid receptors represent a class of transcriptional activators that are able to interact with repressed nucleoprotein templates and recruit necessary activities for chromatin remodeling. The ligand-induced movement of nuclear receptors from inactive states, either in the cytoplasm or in the nucleus, to productive interactions with chromatin is complex and likely reflects the interaction with multiple protein complexes and subcellular structures. Regulation of gene expression by nuclear receptors is thus mediated through the subcellular distribution of inactive receptors, the redistribution of activated receptor complexes to appropriate nuclear domains, the reorganization of chromatin structures for interaction with soluble components of the nucleoplasm, and direct protein-protein contacts between receptors and the basal transcription apparatus.

Animals↗