Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions.
AuthorsGazdik MA, McDonough KA
JournalJ Bacteriol
PubMed ID15805514
'Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that ... More
Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts.
AuthorsPischon N, Darbois LM, Palamakumbura AH, Kessler E, Trackman PC
JournalJ Biol Chem
PubMed ID15138266
'Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation ... More
Control of neuronal size homeostasis by trophic factor-mediated coupling of protein degradation to protein synthesis.
AuthorsFranklin JL, Johnson EM
JournalJ Cell Biol
PubMed ID9732291
'We demonstrate that NGF couples the rate of degradation of long-lived proteins in sympathetic neurons to the rate of protein synthesis. Inhibiting protein synthesis rate by a specific percentage caused an almost equivalent percentage reduction in the degradation rate of long-lived proteins, indicating nearly 1:1 coupling between the two processes. ... More
Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity.
AuthorsPoliakov E, Gentleman S, Cunningham FX, Miller-Ihli NJ, Redmond TM
JournalJ Biol Chem
PubMed ID15951442
'Alignment of sequences of vertebrate beta-carotene 15,15''-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are ... More
Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.
AuthorsNazarenko DA, Dertinger SD, Gasiewicz TA
JournalBiotechniques
PubMed ID11314260
'beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form ... More
Purification and reconstitution of the vacuolar H+-ATPases from lemon fruits and epicotyls.
AuthorsMüller ML, Irkens-Kiesecker U, Kramer D, Taiz L
JournalJ Biol Chem
PubMed ID9139735
'The vacuolar H+-ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H+-transport activities of the purified, reconstituted V-ATPases of ... More
A simple, rapid method for demonstrating bacterial flagella.
AuthorsGrossart HP, Steward GF, Martinez J, Azam F
JournalAppl Environ Microbiol
PubMed ID10919832
'We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well ... More
Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection.
AuthorsLiu Z, Pawliszyn J
JournalAnal Chem
PubMed ID14674468
'A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, ... More
Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.
AuthorsAlonso JL, Mascellaro S, Moreno Y, Ferrús MA, Hernández J
JournalAppl Environ Microbiol
PubMed ID12324366
We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane ... More
Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling.
AuthorsLiu Y, Foote RS, Jacobson SC, Ramsey RS, Ramsey JM
JournalAnal Chem
PubMed ID11028618
Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near ... More
Adhesion of single bacterial cells in the micronewton range.
AuthorsTsang PH, Li G, Brun YV, Freund LB, Tang JX
JournalProc Natl Acad Sci U S A
PubMed ID16585522
The adhesion of bacteria to surfaces plays critical roles in the environment, disease, and industry. In aquatic environments, Caulobacter crescentus is one of the first colonizers of submerged surfaces. Using a micromanipulation technique, we measured the adhesion force of single C. crescentus cells attached to borosilicate substrates through their adhesive ... More
Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies.
Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined ... More
Quantitation of protein.
AuthorsNoble JE, Bailey MJ,
JournalMethods Enzymol
PubMed ID19892168
The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of ... More
Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides.
AuthorsBerezovski MV, Musheev MU, Drabovich AP, Jitkova JV, Krylov SN
JournalNat Protoc
PubMed ID17406423
Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves ... More
The Pro335 --> Leu polymorphism of type 3 inositol 1,4,5-trisphosphate receptor found in mouse inbred lines results in functional change.
AuthorsKim S, Ahn T, Park C
JournalJ Biol Chem
PubMed ID15890645
Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> ... More
A whole new way of looking at things: the use of Dark Reader technology to detect fluorophors.
AuthorsSeville M
JournalElectrophoresis
PubMed ID11332748
The Dark Reader optical system (Clare Chemical Research, Denver, CO, USA) uses relatively low intensity broad-band visible blue light in combination with broad-band optical filters to detect fluorescence with a level of sensitivity that often surpasses that of UV transilluminators and can rival that of laser-based scanners. Applications of DR ... More
The ligand affinity of proteins measured by isothermal denaturation kinetics.
AuthorsEpps DE, Sarver RW, Rogers JM, Herberg JT, Tomich PK
JournalAnal Biochem
PubMed ID11319816
An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the ... More
Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.
AuthorsJones LJ, Haugland RP, Singer VL
JournalBiotechniques
PubMed ID12703310
We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a ... More
Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation.
AuthorsDecreux A, Messiaen J
JournalPlant Cell Physiol
PubMed ID15769808
Wall-associated kinase 1 (WAK1) is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell walls. In order to characterize further the interaction of WAK1 with pectin, a 564 bp DNA sequence corresponding to amino acids 67-254 ... More
Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage.
AuthorsMarathe GK, Johnson C, Billings SD, Southall MD, Pei Y, Spandau D, Murphy RC, Zimmerman GA, McIntyre TM, Travers JB
JournalJ Biol Chem
PubMed ID16115894
Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to ... More
Escherichia coli behavior in the presence of organic matter released by algae exposed to water treatment chemicals.
When exposed to oxidation, algae release dissolved organic matter with significant carbohydrate (52%) and biodegradable (55 to 74%) fractions. This study examined whether algal organic matter (AOM) added in drinking water can compromise water biological stability by supporting bacterial survival. Escherichia coli (1.3 x 10(5) cells ml(-1)) was inoculated in ... More
Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool. Nanomolar human serum albumin quantitation.
AuthorsHarvey MD, Bablekis V, Banks PR, Skinner CD
JournalJ Chromatogr B Biomed Sci Appl
PubMed ID11339278
The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent ... More
Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.
AuthorsParsons SF, Lee G, Spring FA, Willig TN, Peters LL, Gimm JA, Tanner MJ, Mohandas N, Anstee DJ, Chasis JA
JournalBlood
PubMed ID11133776
Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present ... More
Novel chemoautotrophic endosymbiosis between a member of the Epsilonproteobacteria and the hydrothermal-vent gastropod Alviniconcha aff. hessleri (Gastropoda: Provannidae) from the Indian Ocean.
AuthorsSuzuki Y, Sasaki T, Suzuki M, Nogi Y, Miwa T, Takai K, Nealson KH, Horikoshi K
JournalAppl Environ Microbiol
PubMed ID16151136
The hydrothermal-vent gastropod Alviniconcha aff. hessleri from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage ... More
The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase.
AuthorsKöhler GA, Gong X, Bentink S, Theiss S, Pagani GM, Agabian N, Hedstrom L
JournalJ Biol Chem
PubMed ID15665003
Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to ... More
The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid.
AuthorsStieglitz K, Seaton B, Roberts MF
JournalJ Biol Chem
PubMed ID10585404
The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the ... More
Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling.
AuthorsJin LJ, Giordano BC, Landers JP
JournalAnal Chem
PubMed ID11681478
The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible ... More
Cytokine profiling of macrophages exposed to Porphyromonas gingivalis, its lipopolysaccharide, or its FimA protein.
AuthorsZhou Q, Desta T, Fenton M, Graves DT, Amar S
JournalInfect Immun
PubMed ID15664935
To characterize the roles of Porphyromonas gingivalis and its components in the disease processes, we investigated the cytokine profile induced by live P. gingivalis, its lipopolysaccharides (LPS), and its major fimbrial protein, fimbrillin (FimA). Using cytokine antibody arrays, we found that P. gingivalis LPS and FimA induced a similar profile ... More