Search PubMed⌕ Search

Biomedical subjects

S Chu

Publications and source records attributed to S Chu.

At least 19 recordsLinked to original sources
PubMed1999

Single-polymer dynamics in steady shear flow.

The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased.

Bacteriophage lambda↗
PubMed1999

Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3.

Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.

Aging↗
PubMed1999

Keratinocyte growth factor stimulates CLC-2 expression in primary fetal rat distal lung epithelial cells.

Keratinocyte growth factor (KGF) is mitogenic for epithelial cells and induces cystic dilation of fetal lung explants through cystic fibrosis transmembrane conductance regulator-independent chloride channels. One candidate fetal lung chloride channel that is highly expressed on the apical surface of the respiratory epithelium and markedly downregulated after birth is CLC-2. We hypothesized that KGF regulates CLC-2 expression in the fetal lung. Primary fetal rat distal lung epithelial cell monolayers were grown in medium containing 10 ng/ml KGF for 48 h. CLC-2 protein was increased by Western blot analysis of whole-cell lysates in KGF-treated cultures. Similarly, KGF stimulated CLC-2 messenger RNA (mRNA) by Northern blot analysis. This enhanced expression was dose-dependent and maximal at 48 h with 10 ng/ml KGF. Promoter-reporter gene experiments demonstrated that KGF did not stimulate gene transcription. By inhibition of new mRNA synthesis with actinomycin D, evidence was obtained that KGF stabilizes CLC-2 mRNA. We speculate that KGF may positively influence pulmonary chloride and fluid secretion by a secondary pathway affecting CLC-2 degradation.

Animals↗
PubMed1999

Dynamic regulation of gastric surface pH by luminal pH.

In vivo confocal imaging of the mucosal surface of rat stomach was used to measure pH noninvasively under the mucus gel layer while simultaneously imaging mucus gel thickness and tissue architecture. When tissue was superfused at pH 3, the 25 microm adjacent to the epithelial surface was relatively alkaline (pH 4.1 +/- 0.1), and surface alkalinity was enhanced by topical dimethyl prostaglandin E2 (pH 4.8 +/- 0.2). Luminal pH was changed from pH 3 to pH 5 to mimic the fasted-to-fed transition in intragastric pH in rats. Under pH 5 superfusion, surface pH was relatively acidic (pH 4.2 +/- 0.2). This surface acidity was enhanced by pentagastrin (pH 3.5 +/- 0.2) and eliminated by omeprazole, implicating parietal cell H,K-ATPase as the dominant regulator of surface pH under pH 5 superfusion. With either pH 5 or pH 3 superfusion (a) gastric pit lumens had the most divergent pH from luminal superfusates; (b) qualitatively similar results were observed with and without superfusion flow; (c) local mucus gel thickness was a poor predictor of surface pH values; and (d) no channels carrying primary gastric gland fluid through the mucus were observed. The model of gastric defense that includes an alkaline mucus gel and viscous fingering of secreted acid through the mucus may be appropriate at the intragastric pH of the fasted, but not fed, animal.

Animals↗
PubMed1998

The transcriptional program of sporulation in budding yeast.

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

Animals↗
PubMed1998

Restricted tissue expression pattern of a novel human rasGAP-related gene and its murine ortholog.

The mammalian rasGAPs constitute a group of widely expressed proteins involved in the negative regulation of ras-mediated signaling. In this study we have isolated a novel human gene, RASAL (Ras GTPase-activating-like) and its murine ortholog, MRASAL which are most similar to the GAP1 family of rasGAP proteins, based upon the presence and organization of specific conserved domains. Full-length human and murine mRNA sequences are predicted to encode 804 and 799 amino acid polypeptides, respectively. Sequence analysis of these two proteins revealed the presence of two N-terminal calcium-dependent phospholipid binding C2 domains, a conserved GAP related domain (GRD) and a C-terminal pleckstrin homology (PH) domain. Northern blot and mRNA in situ hybridization analyses indicate that RASAL, in contrast to other mammalian rasGAP proteins, has a limited expression pattern; RASAL is highly expressed in the follicular cells of the thyroid and the adrenal medulla and expressed at lower levels in brain, spinal cord and trachea. Human RASAL has been localized by radiation hybrid mapping to chromosome 12q23-24.

Amino Acid Sequence↗
PubMed1998

Response of flexible polymers to a sudden elongational flow

Individual polymers at thermal equilibrium were exposed to an elongational flow producing a high strain rate, and their dynamics were recorded with video fluorescence microscopy. The flow was turned on suddenly so that the entire evolution of molecular conformation could be observed without initial perturbations. The rate of stretching of individual molecules is highly variable and depends on the molecular conformation that develops during stretching. This variability is due to a dependence of the dynamics on the initial, random equilibrium conformation of the polymer coil. The increasing appearance at high strain rates of slowly unraveling hairpin folds is an example of nonergodic dynamics, which can occur when a statistical mechanical system is subjected to nonadiabatic, or "sudden," external forces.

Journal Article↗
PubMed1998

Quantitation of TNP-470 and its metabolites in human plasma: sample handling, assay performance and stability.

A selective and sensitive assay for the determination of TNP-470 and two of its metabolites, AGM-1883 and M-II, in human plasma was developed. The assay involved liquid-liquid extraction followed by analysis using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Because TNP-470 is most stable in a pH of 4-5, an acidification procedure was utilized to prevent degradation of TNP-470 during sample collection which involved acidifying the whole blood sample collected with 5 mg of citric acid per ml of blood. Liquid-liquid extraction using an organic solvent mixture was chosen over solid-phase extraction to minimize the degradation of TNP-470 during solvent evaporation.

Acetamides↗
PubMed1998

Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80.

Gametogenesis requires the successful coordination of two key processes, meiotic nuclear division and gamete morphogenesis. A central regulatory step in progression through gametogenesis occurs at the pachytene stage of meiotic prophase. We find that Ndt80 functions at pachytene of yeast gametogenesis (sporulation) to activate transcription of a set of genes required for both meiotic division (e.g., B-type cyclins) and gamete formation (e.g., SPS1). Ectopic synthesis of Ndt80 in vegetative cells induces transcription of these genes, and recombinant Ndt80 protein binds to a conserved sequence in their upstream region. Transcription of NDT80 itself is dependent on Ime1, which activates expression of early sporulation genes. Transcription of the Ndt80-regulated gene CLB1 is mediated by the checkpoint gene RAD17. Thus Ndt80 is a pivotal component of a transcriptional cascade programming yeast gametogenesis and may also be a target of meiotic checkpoint control.

Cyclin B↗
PubMed1998

Improving clinical pathway design: lessons learned from a computerised prototype.

Increasing costs of health care, fuelled by demand for high quality, cost-efficient health care has propelled hospitals to restructure their patient care delivery systems. One such effort is the adaptation of an engineering project management methodology, the critical path method (CPM), as a tool to organise, standardise and improve the quality of healthcare delivery and hence patient outcomes. However, the two-dimensional nature and the size of paper impose severe limitations on the manual clinical pathways currently in use by hospitals. This paper analyses these inherent limitations and discusses some of the problems encountered in an attempt in early 1996 to create an electronic care map planner (CMP) based on the precedence diagramming method (PDM) model. It also reports on a current project to create a computerised clinical pathway tool to resolve the identified problems.

Cost-Benefit Analysis↗
PubMed1998

Structural determinants of cortisol resistance in the guinea pig glucocorticoid receptor.

The guinea pig exhibits resistance to glucocorticoids in vivo which results from the guinea pig glucocorticoid receptor (GR) having a lower affinity for cortisol than the human GR. Cloning of the guinea pig GR has revealed that the amino acid sequence of the ligand-binding domain (LBD) differs from the human GR at 24 residues. The present study confirms that the decreased sensitivity and binding affinity of the guinea pig GR are conferred in vitro by the LBD. Further, the substitutions in the LBD do not confer altered relative steroid sensitivity or selectivity compared with the human GR. The altered sensitivity and binding of dexamethasone are confined to the first third of the LBD, which contains 5 nonconservative substitutions in a region that is otherwise highly conserved across several species of GR. These residues, either alone or in combination, were targeted for site-directed mutagenesis in both the human and guinea pig LBD. Trans-activation studies with these mutant GR failed to exclusively implicate or exclude any of the residues in the observed resistance. Rather, the changes, with 1 exception, caused a decrease in sensitivity, suggesting that critical intramolecular interactions involving at least 4 of these residues determine the correct conformation of this region. Recent molecular modeling of the GR LBD structure suggests that although the above region is not part of the core ligand-binding pocket, it is required to maintain the conformation of the binding pocket.

Amino Acid Sequence↗
PubMed1997

Alternative mRNA splice variants of the rat ClC-2 chloride channel gene are expressed in lung: genomic sequence and organization of ClC-2.

The ClC-2 epithelial cell chloride channel is a voltage-, tonicity- and pH-regulated member of the ClC super family. We have previously shown that rat lung ClC-2 (rClC-2) is down-regulated at birth, and molecular diversity is generated by alternative splicing [Murray et al. (1995) Am. J. Respir. Cell Mol. Biol. 12, 597-604; Murray et al. (1996) Am. J. Physiol. 271, L829-L837; Chu et al . (1996) Nucleic Acids Res. 24, 3453-3457]. To investigate other possible mRNA splice variations, we sequenced the entire rClC-2 gene and found that ClC-2Sa (formerly ClC-2S) results from the deletion of exon 20. The preceding intron 19 has an unusually high CT content and a rare AAG acceptor site. Because both features were also found in intron 13, we next tested the hypothesis that intron 13 would be involved in alternative splicing. As predicted, a second splice product, ClC-2Sb, was found by RT-PCR, but only in lung. When we compared the genomic maps of rClC-2 and human ClC-1 (hClC-1), striking similarities were found in each exon except for rClC-2 exon 20, which is absent in hClC-1. These observations suggest that ClC-1 and ClC-2 may have evolved by gene duplication, mutation and DNA rearrangement.

Alternative Splicing↗
PubMed1997

Identification of a splice variant of the rat estrogen receptor beta gene.

Recently a second estrogen receptor termed estrogen receptor beta (ERbeta) has been cloned and characterized, and shown to be expressed at the highest levels in ovarian granulosa cells and prostatic epithelium. In the course of amplifying a region of the ligand-binding domain of the rat ERbeta cDNA we identified a second, larger transcript which appears to arise through differential splicing. The second isoform has 54 nucleotides inserted after position 1372 encoding 18 additional amino acids. Both isoforms are expressed at similar relative abundance in a range of tissues.

Amino Acid Sequence↗
PubMed1997

Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration.

Physicochemical properties of the cationic liposomes, including structure of the cationic lipid-to-DNA ratio, liposome particle size, and inclusion of the helper lipids, were studied for their effect on the level, site, and duration time of gene expression in vivo by intravenous administration. Using a cytomegalovirus (CMV)-driven gene expression system containing either the luciferase or green fluorescence protein gene as a reporter and two cationic lipids [N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP)], we demonstrated in vivo by a single intravenous injection of DNA/liposome complexes into mice, that cationic liposomes are capable of transfecting cells in organs such as the lung, heart, liver, spleen, and kidney. Transfection efficiency is determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA. Although the presence of cholesterol in DOTAP liposomes did not affect transfection activity, inclusion of dioleoylphosphatidylethanolamine (DOPE) into either DOTAP or DOTMA liposomes significantly decreases liposome transfection activity in vivo. Results form time course show that gene expression in different organs is transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4. Experiments with repeated injections showed that the peak level of gene expression could be regained by subsequent injection.

Animals↗
PubMed1997

The dynamics of partially extended single molecules of DNA.

The behaviour of an isolated polymer floating in a solvent forms the basis of our understanding of polymer dynamics. Classical theories describe the motion of a polymer with linear equations of motion, which yield a set of 'normal modes', analogous to the fundamental frequency and the harmonics of a vibrating violin string. But hydrodynamic interactions make polymer dynamics inherently nonlinear, and the linearizing approximations required for the normal-mode picture have therefore been questioned. Here we test the normal-mode theory by measuring the fluctuations of single molecules of DNA held in a partially extended state with optical tweezers. We find that the motion of the DNA can be described by linearly independent normal modes, and we have experimentally determined the eigenstates of the system. Furthermore, we show that the spectrum of relaxation times obeys a power law.

Biopolymers↗
PubMed1997

Single polymer dynamics in an elongational flow.

The stretching of individual polymers in a spatially homogeneous velocity gradient was observed through use of fluorescently labeled DNA molecules. The probability distribution of molecular extension was determined as a function of time and strain rate. Although some molecules reached steady state, the average extension did not, even after a approximately 300-fold distortion of the underlying fluid element. At the highest strain rates, distinct conformational shapes with differing dynamics were observed. There was considerable variation in the onset of stretching, and chains with a dumbbell shape stretched more rapidly than folded ones. As the strain rate was increased, chains did not deform with the fluid element. The steady-state extension can be described by a model consisting of two beads connected by a spring representing the entropic elasticity of a worm-like chain, but the average dynamics cannot.

Bacteriophage lambda↗
PubMed1997

Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy.

This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multiwalled carbon nanotubes as tapping-mode supertips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities. One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbenemediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyl-diazirinyl crosslinker. We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates.

Alkanes↗