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Biomedical subjects

L He

Publications and source records attributed to L He.

At least 19 recordsLinked to original sources
PubMed2003

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser.

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Bacterial Proteins↗
PubMed2002

Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods.

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.

Animals↗
PubMed2002

Carbonate apatite coating on titanium induced rapidly by precalcification.

Chemical treatments have been thought to be promised methods for improving bioactivity of titanium. In this work, the effect of precalcification with boiling saturated Ca(OH)2 solution on bioactivation of titanium was investigated. After precalcification and soaking in supersaturated Ca-P solution (SCP), calcium phosphate rapidly precipitated onto the surfaces of titanium, and after only three days an uniform apatite layer was found up to thickness of a few micrometers. The observation using scanning electron microscopy (SEM) showed that the coating was composed of a number of small crystal grains. The investigation by X-ray energy dispersion spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) indicated that the coating was Ca-deficient carbonate apatite. Based on the analyses for the surfaces and SCP, a mechanism of precipitation of apatite was proposed in thermal dynamics and kinetics.

Apatites↗
PubMed2002

A family-based and case-control association study of the NOTCH4 gene and schizophrenia.

Recently a strong positive association between schizophrenia and Notch4 has been reported. Both individual markers and haplotypes showed association with the disease, with five markers (three microsatellites and two SNPs) being tested. In order to test this finding we genotyped these markers in the Han Chinese population using a sample of 544 cases and 621 controls as well as >300 trios. Analysis of allele, genotype and haplotype frequencies in both samples showed no association between the markers and the disease. Our results would indicate that a significant role for the Notch4 gene in schizophrenia can be ruled out in the Han Chinese. However, similar studies are necessary in the Caucasian population as linkage disequilibrium arrangements and founder effects may differ between these two populations.

Adolescent↗
PubMed2002

Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice.

Various heme-containing proteins have been proposed as primary molecular O(2) sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91(phox)) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O(2)-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K(+) currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca(2+) concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91(phox) gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O(2) sensor in arterial chemoreception.

Animals↗
PubMed2002

Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia.

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.

Adaptation, Physiological↗
PubMed2002

Chronic hypoxia upregulates connexin43 expression in rat carotid body and petrosal ganglion.

Recent studies have demonstrated that oxygen-sensitive type I cells in the carotid body express the gap junction-forming protein connexin43 (Cx43). In the present study, we examined the hypothesis that chronic exposure to hypoxia increases Cx43 expression in type I cells as well as in chemoafferent neurons in the petrosal ganglion. Immunocytochemical studies in tissues from normal rats revealed diffuse and granular Cx43-like immunoreactivity in the cytoplasm of type I cells and dense punctate spots of immunoreactive product at the margins of type I cells and near the borders of chemosensory cell lobules. Cx43-like immunoreactivity was not detectable in petrosal ganglion neurons from normal animals. After a 2-wk exposure to hypobaric (380 Torr) hypoxia, Cx43 immunostaining was substantially enhanced in and around type I cells. Moreover, chronic hypoxia elicited the expression of Cx43-like immunoreactivity in the cytoplasm of afferent neurons throughout the petrosal ganglion. Quantitative RT-PCR studies indicate that chronic hypoxia evokes a substantial increase in Cx43 mRNA levels in the carotid body, along with a marked elevation of Cx43 expression in the petrosal ganglion. Increased Cx43 expression and gap junction formation in type I cells and sensory neurons may contribute to carotid body adaptation during sustained stimulation in extreme physiological conditions.

Actins↗
PubMed2002

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.

Animals↗
PubMed2001

Submicrometer metallic barcodes.

We synthesized multimetal microrods intrinsically encoded with submicrometer stripes. Complex striping patterns are readily prepared by sequential electrochemical deposition of metal ions into templates with uniformly sized pores. The differential reflectivity of adjacent stripes enables identification of the striping patterns by conventional light microscopy. This readout mechanism does not interfere with the use of fluorescence for detection of analytes bound to particles by affinity capture, as demonstrated by DNA and protein bioassays.

Animals↗
PubMed2001

Determination of cell viability in single or mixed samples using capillary electrophoresis laser-induced fluorescence microfluidic systems.

The advent of high-efficiency microbial separations will have a profound effect on both chemistry and microbiology. For the first time, it appears that it may be possible to obtain qualitative and quantitative information on microbial systems with the accuracy, precision, speed, and throughput that currently is found for chemical systems. Recently it was suggested that an analytical separations-based approach for determining the viability of cells would be advantageous. The feasibility of such an approach is demonstrated using CE-LIF of two bacteria and yeast. The analytical procedures and figures of merit are outlined. High-throughput analyses and evaluation of microorganisms now appear to be possible.

Bifidobacterium↗
PubMed2001

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.

Animals↗
PubMed2001

Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry.

Room-temperature ionic liquids are useful as solvents for organic synthesis, electrochemical studies, and separations. We wished to examine whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI. Several different ionic matrixes were synthesized and tested, using peptides, proteins, and poly(ethylene glycol) (PEG-2000). All ionic liquids tested have excellent solubilizing properties and vacuum stability compared to other commonly used liquid and solid matrixes. However, they varied widely in their ability to produce analyte gas-phase ions. Certain ionic matrixes, however, produce homogeneous solutions of greater vacuum stability, higher ion peak intensity, and equivalent or lower detection limits than currently used solid matrixes. Clearly, ionic liquids and their more amorphous solid analogues merit further investigation as MALDI matrixes.

Animals↗
PubMed2001

Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells.

Vinca alkaloids are used extensively in the treatment of childhood acute lymphoblastic leukemia (ALL) and despite their usefulness, drug resistance remains a serious clinical problem. Vinca alkaloids bind to the beta-tubulin subunit of the alpha/beta-tubulin heterodimer and inhibit polymerization of microtubules. Recent studies have implicated altered beta-tubulin isotype expression and mutations in resistance to microtubule-stabilizing agents. Microtubule-associated protein (MAP) MAP4 binds to and stabilizes microtubules, and increased expression is associated with decreased sensitivity to microtubule-depolymerizing agents. To address the significance of beta-tubulin and MAP4 alterations in childhood ALL, two CCRF-CEM-derived Vinca alkaloid resistant cell lines, VCR R (vincristine) and VLB100 (vinblastine), were examined. Decreased expression of class III beta-tubulin was detected in both VCR R and VLB100 cells. VCR R cells and to a lesser extent VLB100 cells expressed increased levels of MAP4 protein. Increased microtubule stability was observed in these VCR R cells as identified by the high levels of polymerized tubulin (45.6 +/- 2.6%; P < 0.005) compared with CEM and VLB100 cells (24.7 +/- 3.3% and 24.7 +/- 2.5%, respectively). Expression was associated with a single MAP4 isoform in the polymerized microtubule fraction in CEM and VCR cells. In contrast, VLB100 cells expressed a lower molecular weight isoform in the polymerized fraction. Two-dimensional-PAGE and immunoblotting revealed marked posttranslational changes in class I beta-tubulin in VCR R cells not evident in CEM cells. Sequencing of the beta-tubulin (HM40) gene identified a point mutation in VCR R cells in nucleotide 843 (CTC-->ATC; Leu(240)-->Ile) that was not present in CEM or VLB100 cells. This mutation resides in a region of beta-tubulin that lies in close proximity to the alpha/beta tubulin interface. Multiple alterations related to normal microtubule function were identified in ALL cells selected for resistance to Vinca alkaloids, and these alterations may provide important insight into mechanisms mediating resistance to Vinca alkaloids.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin.

A monoclonal IgG isolated from a patient with multiple myeloma has been shown to bind to exosite II of thrombin, prolong both the thrombin time and the activated partial thromboplastin time (aPTT) when added to normal plasma, and alter the kinetics of hydrolysis of synthetic peptide substrates. Although the IgG does not affect cleavage of fibrinogen by thrombin, it increases the rate of inhibition of thrombin by purified antithrombin approximately 3-fold. Experiments with plasma immunodepleted of antithrombin or heparin cofactor II confirm that prolongation of the thrombin time requires antithrombin. By contrast, prolongation of the aPTT requires neither antithrombin nor heparin cofactor II. The IgG delays clotting of plasma initiated by purified factor IXa but has much less of an effect on clotting initiated by factor Xa. In a purified system, the IgG decreases the rate of activation of factor VIII by thrombin. These studies indicate that binding of a monoclonal IgG to exosite II prolongs the thrombin time indirectly by accelerating the thrombin-antithrombin reaction and may prolong the aPTT by interfering with activation of factor VIII, thereby diminishing the catalytic activity of the factor IXa/VIIIa complex.

Anions↗
PubMed2001

Highly enantioselective synthesis of alpha,beta-diaminopropanoic acid derivatives using a catalytic asymmetric hydrogenation approach.

Rh-DuPhos-catalyzed asymmetric hydrogenation of alpha,beta-diamidoacrylates provides a highly efficient and enantioselective route to chiral alpha,beta-diaminopropanoic acid derivatives. The mechanistic course of the hydrogenation was studied using isotopically enriched enamide complexes and phosphorus and carbon NMR. Addition of methyl alpha-N-benzoyl-beta-N-acetyl-diaminopropenoate to the solvated catalyst gave a single 1:1 enamide complex and demonstrated the binding of the olefin and alpha-amide carbonyl group; the carboxylate and beta-N-acyl groups did not bind to the metal. Changes to the electronic and steric properties of the beta-N-acyl group were well tolerated; however, small changes to the binding alpha-N-acyl group were found to significantly affect hydrogenation yields.

Journal Article↗
PubMed2001

Expedient asymmetric synthesis of all four isomers of N,N'-protected 2,3-diaminobutanoic acid.

2,3-Diaminobutanoic acid (DAB) is found in several peptide antibiotics, toxins, and biologically active molecules. This paper describes the practical and highly enantioselective synthesis of all four N,N'-protected DAB stereoisomers using an asymmetric Rh(I)-phosphine-catalyzed hydrogenation of isomeric enamides as the key step. Thermal and photochemical isomerization of the enamide hydrogenation substrates coupled with catalyst-geometric isomer pairing allows targeted synthesis of single DAB isomers in maximum yield.

Journal Article↗