Biomedical subjects

B He

Publications and source records attributed to B He.

At least 19 recordsLinked to original sources

PubMed1999

Microfabricated filters for microfluidic analytical systems.

Solvent and reagent filters were micromachined into quartz wafers using deep reactive ion etching to create a network of intersecting 1.5 x 10 microns channels. When placed at the bottom of reservoirs with a side exit, this channel network behaved as a lateral percolation filter composed of an array of cubelike structures one layer deep. Flow through these filters was driven by electroosmotic flow (EOF). Silanol groups at the walls of channels in the network provided the requisite charge to trigger EOF when voltage was applied laterally to the filter. Adsorption of cationic proteins in this silanol-rich matrix was controlled by the application of a polyacrylamide coating prepared by bonding N-hydroxysuccinimide (NHS)-activated poly(acrylic acid) to (gamma-aminopropyl)silane-derivatized filters. Subsequent reaction of residual NHS groups in the coating with 2-(2-aminoethoxy)ethanol provided channels of low charge density and adsorptivity. These lateral percolation filters were shown to be efficacious in filtering solvents containing a variety of particulate materials, ranging from dust to cells.

Adsorption↗

PubMed1999

Parathyroid hormone-related protein interacts with RNA.

Parathyroid hormone-related protein (PTHrP) is a secreted protein that acts as an autocrine and paracrine mediator of cell proliferation and differentiation. In addition to its biological activity that is mediated through signal transduction cascades, there is evidence for an intracellular role for PTHrP in cell cycle progression and apoptosis. These effects are mediated through a mid-region nuclear targeting sequence (NTS) that localizes PTHrP to the region of the nucleolus where ribonucleoprotein complexes form in vivo. In this work, we show that endogenous, transfected, and in vitro translated PTHrP proteins bind homopolymeric and total cellular RNAs at salt concentrations up to 1 M. A peptide representing the PTHrP NTS was effective in competing with the wild-type protein for RNA binding, whereas a similar peptide representing the nucleolin NTS was not. Site-directed mutagenesis revealed that the binding of PTHrP to RNA was direct and was dependent on preservation of a core GXKKXXK motif, embedded in the PTHrP NTS, which is shared with other RNA-binding proteins. The current observations are the first to document RNA binding by a secreted cellular protein and predict a role for PTHrP in regulating RNA metabolism that may be related to its localization in the nucleolus of cells in vivo.

Amino Acid Sequence↗

PubMed1999

The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell-adhesion signaling.

The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH-fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.

Animals↗

PubMed1999

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets.

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.

Adenylyl Cyclases↗

PubMed1999

Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life science.

Liquid chromatography and electrophoresis played a major role in the life-science revolution, most strikingly in protein purification, peptide fractionation and sequencing, amino acid analysis, and DNA sequencing. The objective of this article is to examine the potential role of separation systems in the continuing evolution of biochemistry, biotechnology and molecular biology. Very small chip-based systems may change how chemical analyses in biology, medical research and health care evolve over the next decade.

Biological Science Disciplines↗

PubMed1999

Yield of DNA strand breaks after base oxidation of plasmid DNA.

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.

DNA Damage↗

PubMed1998

Cytokine and chemokine mRNA expressing cells in muscle tissues of experimental autoimmune myasthenia gravis.

In-situ hybridization with labeled oligonucleotide probes was applied to explore cytokine and chemokine mRNA expression in sections of striated muscle, the target organ in experimental autoimmune myasthenia gravis (EAMG), induced in Lewis rats by immunization with acetylcholine receptor (AChR) and complete Freund's adjuvant (CFA). A transient burst of TNF-alpha, IL-1beta and IL-6 mRNA expressing cells was detected during the early phase of EAMG. This cytokine pattern was related to muscular infiltration of macrophages. Levels of IL-4, IL-10, IFN-gamma, cytolysin and TGF-beta mRNA expressing cells were low and observed mainly during the early phase of EAMG. C-C chemokine RANTES, MCP, MIP-1alpha and MIP-2 mRNA expressing cells were not detected over the course of EAMG. The low and transient expression of cytokines in EAMG muscle tissues suggests that the immune effector responses are unlikely operated by infiltrating cells in muscle. Muscular infiltrations in EAMG are unlikely due to local accumulation of C-C chemokines.

Animals↗

PubMed1998

The paramyxovirus SV5 small hydrophobic (SH) protein is not essential for virus growth in tissue culture cells.

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.

Animals↗

PubMed1998

Fabrication of nanocolumns for liquid chromatography.

This paper shows that in situ micromachining can be used to simultaneously position and define (i) support particles, (ii) convective transport channels, (iii) an inlet distribution network of channels, and (iv) outlet channels in multiple chromatography columns on a single quartz wafer to the level of a few tenths of a micrometer. Stationary phases were bonded to 5 x 5 x 10 microns collocated monolith support structures separated by rectangular channels 1.5 microns wide and 10 microns deep with a low degree of deviation of channel width between the top and bottom of channels. High aspect ratio microfabrication can only be achieved with deep reactive ion etching. The volume of a 150 microns x 4.5 cm column was 18 nL. Column efficiency was evaluated in the capillary electrochromatography (CEC) mode using rhodamine 123 and a hydrocarbon stationary phase. Plate heights in these columns were typically 0.6 micron in the nonretained and 1.3 microns in the retained modes of operation. Columns were designed to have identical mobile-phase velocity in all channels in an effort to minimize outgassing during operation. When the total lateral cross-sectional area of channels at all points along the separation axis is identical, linear velocity of the mobile phase in a CEC column should be the same. Columns were operated at atmospheric pressure.

Chromatography, Liquid↗

PubMed1998

Microfabricated liquid chromatography columns based on collocated monolith support structures.

There is great interest today in massively parallel analytical strategies as a way to accelerate the rate of discovery in biological research; among them being 'biochips' and 'laboratories-on-a-chip'. The concept in the 'chip' approach is that minaturization will allow large numbers of operations to be performed in parallel in a small space, as in electronics. Proceeding with the semiconductor analogy, this paper demonstrates that in situ micromachining can be used to simultaneously fabricate millions of micrometer size, particle like structures in multiple liquid chromatography columns on a single wafer. Reduction of this widely used bioanalytical tool to the nanoliter volume, parallel processing, chip format is a significant step toward laboratories-on-a-chip.

Chemistry Techniques, Analytical↗

PubMed1998

Calculation of concentrations of equilibrium components in an in vitro activity test of vancomycin antibiotics and the possible mode of action.

The vancomycin group of antibiotics is considered to act by binding the bacterial cell wall mucopeptide precursor terminating in -L-Lys-D-Ala-D-Ala. The dimerization of these antibiotics is also believed to play a role in the action. In this paper, we analyzed the equilibria in the in vitro antibacterial activity test of the vancomycin antibiotics both with and without the cell wall precursor analogue di-acetyl-L-Lys-D-Ala-D-Ala (DALAA). Based on the equilibria and concentration balance, we obtained 10 equations (seven quadratic equations and three linear equations) containing 10 equilibrium concentrations which relate to the antibiotic, cell wall precursor and DALAA. A computer program was written to solve these equations from known dimerization constant and the binding constants (both monomer and dimer) with DALAA of the antibiotic. The concentrations in the test for vancomycin and eremomycin were obtained. The antibiotic activity of these antibiotics may be quantitatively correlated with their dimerization constants and the binding constants through the calculation. By analyzing the calculated results, we concluded that the cell wall-bound dimer may be the major contributor to the antibiotic activity in the case of eremomycin, while the cell wall-bound monomer is possibly the determinant for the activity of vancomycin.

Anti-Bacterial Agents↗

PubMed1998

The gamma134.5 protein of herpes simplex virus 1 has the structural and functional attributes of a protein phosphatase 1 regulatory subunit and is present in a high molecular weight complex with the enzyme in infected cells.

The carboxyl-terminal domain of the gamma134.5 protein of the herpes simplex virus 1 binds to protein phosphatase 1alpha (PP1) and is required to prevent the shut-off of protein synthesis resulting from phosphorylation of the alpha subunit of eIF-2 by the double-stranded RNA-activated protein kinase. The corresponding domain of the conserved GADD34 protein homologous to gamma134.5 functionally substitutes for gamma134.5. This report shows that gamma134.5 and PP1 form a complex in the infected cells, that fractions containing this complex specifically dephosphorylate eIF-2alpha, and that both gamma134.5 and GADD34 proteins contain the amino acid sequence motif common to subunits of PP1 that is required for binding to the PP1 catalytic subunit. An oligopeptide containing this motif competes with gamma134.5 for binding to PP1. Substitution of Val193 and Phe195 in the PP1-binding motif abolished activity. These results suggest that the carboxyl-terminal domain of gamma134.5 protein has the structural and functional attributes of a subunit of PP1 specific for eIF-2alpha, that it has evolved to preclude shut-off of protein synthesis, and that GADD34 may have a similar function.

Amino Acid Sequence↗

PubMed1998

Characterization of an unusual, sequence-specific termination signal for T7 RNA polymerase.

We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.

Base Sequence↗

PubMed1998

Pausing and termination by bacteriophage T7 RNA polymerase.

Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid. The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results.

Bacteriophage T7↗

PubMed1998

Linkage and association analysis of genes encoding cytokines and myelin proteins in multiple sclerosis.

Several genetic factors are likely to play a role in the etiology of multiple sclerosis (MS). We used a candidate gene strategy in a study of polymorphic markers within or close to genes encoding cytokines (interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-4 receptor (IL-4R), IL-10, transforming growth factor-beta1 and -beta2) and myelin proteins (2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNP:ase), myelin associated glycoprotein, oligodendrocyte myelin glycoprotein, proteolipid protein) in 34 Swedish multiplex MS families and in 147 sporadic MS patients and 95 healthy controls. No evidence for linkage was observed in two-point linkage analysis. However, a slightly positive LOD score of 0.88 (theta = 0.01) for IFN-gamma was found. Affected pedigree member (APM) analysis indicated a possible linkage with TGF-beta2 (p = 0.008) and IL-4R (p = 0.043). None of the cytokine markers were associated with MS in case-control analysis. Our results suggest a possible importance of the TGF-beta2, IL-4R and IFN-gamma genes in MS.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1998

Promoter specificity determinants of T7 RNA polymerase.

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.

Amino Acid Sequence↗

PubMed1998

Neuropeptide Y antibody attenuates 2-deoxy-D-glucose induced feeding in rats.

2-deoxy-D-glucose (2-DG) has been shown to induce increased feeding responses in animals. Recent studies suggest the possible involvement of neuropeptide Y (NPY) in 2-DG-induced feeding. The present study examined the effect of immunoneutralization of endogenous NPY on 2-DG-induced feeding. NPY antibody injected into the paraventricular nucleus of the rats significantly attenuated 2-DG-induced feeding, suggesting that hypothalamic NPY may mediate, at least partly, the effect of 2-DG on food intake.

Animals↗

PubMed1998

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis.

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell costimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28-/-, CD40L-/-). Compared to wild-type mice, the CD28-/- mice became less susceptible, and CD40L-/- mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28-/- mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28-/- mice. In the CD40L-/- mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28-/- mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.

Amino Acid Sequence↗