Biomedical subjects

J A Cox

Publications and source records attributed to J A Cox.

At least 19 recordsLinked to original sources

PubMed2006

Laser cooling of trapped ytterbium ions with an ultraviolet diode laser.

We demonstrate an ultraviolet diode laser system for cooling of trapped ytterbium ions. The laser power and linewidth are comparable to those of previous systems based on resonant frequency doubling, but the system is simpler, more robust, and less expensive. We use the laser system to cool small numbers of ytterbium ions confined in a linear Paul trap. From the observed spectra, we deduce final temperatures of < 270 mK.

Journal Article↗

PubMed2006

Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X(3) receptor subunit transgenes.

The two paralogous P2X receptor subunit genes p2rx3.1 and p2rx3.2 are selectively expressed in overlapping, but unique, patterns of sensory neurons in the developing zebrafish. We constructed a series of transgenes derived from both genes using the recombineering technique. Transgenes utilizing either enhanced green fluorescent protein or monomeric red fluorescent protein-1 were shown to be expressed with the same spatial and temporal patterns as the native genes. The p2rx3.1-derived transgenes labeled the vast majority of the Rohon-Beard neurons in the spinal cord and neurons of the trigeminal ganglia. The p2rx3.2-derived transgene labeled fewer Rohon-Beard and trigeminal neurons than what was observed for the p2rx3.1-derived transgenes, but was also detected in neurons of the epibranchial ganglia. Three distinct populations of sensory neurons were detected: those expressing only one or the other paralog, and those expressing both paralogs. The fluorescent proteins encoded by the transgenes allowed for visualization of the neuronal somas as well as their peripheral and central projections. These reagents should prove extremely useful in providing the basis for future studies aimed at elucidating the developmental and physiological attributes of sensory neurons.

Animals↗

PubMed2003

Molecular characterization of the zebrafish P2X receptor subunit gene family.

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.

Adenosine Triphosphate↗

PubMed2002

Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration.

Inter-alpha-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.

Animals↗

PubMed2001

Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

Amino Acid Sequence↗

PubMed2001

Carbon electrodes modified with ruthenium metallodendrimer multilayers for the mediated oxidation of methionine and insulin at physiological pH.

A pentaerythritol-based metallodendrimer with ruthenium(II) terpyridine units, Ru(II)Den, catalyzed the oxidation of L-methionine and insulin at pH 7.0. The Ru(II)Den was immobilized on a carbon surface through layer-by-layer electrostatic deposition; the negatively charged polymer, poly(styrene sulfonate), was its counterpart. These bilayers were assembled on a glassy carbon electrode that was first modified by deposition of a layer of the conjugate base of sulfanilic acid and then with quaternized poly(4-vinylpyridine). Reversible voltammetry for the Ru(II/III) redox couple was observed, the current for which increased linearly with layer number, n, of Ru(II)Den up to n = 12. Cyclic voltammetry was used to demonstrate the mediation of L-methionine oxidation by a Ru(II)Den-containing multilayer assembly. Flow injection amperometric determination of insulin at pH 7.0 at this modified electrode yielded a calibration curve with the following characteristics: linear dynamic range, 6 nM-0.4 microM; sensitivity, 225 nA microM(-1); detection limit (k = 3 criterion), 2 nM. Of particular importance was that the sensitivity was proportional to the number of Ru(II)Den layers.

Animals↗

PubMed2001

The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel.

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog alphabeta-methylene ATP(alphabetameATP) (P2X(1) and P2X(3)) and those that are not (P2X(2), P2X(4), P2X(5), P2X(6), and P2X(7)). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the alphabetameATP-sensitive rat P2X(1) subunit with that of the alphabetameATP-insensitive rat P2X(2) subunit resulted in a receptor that was still alphabetameATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X(2) subunit with one from an alphabetameATP-sensitive subunit (either rat P2X(1) or P2X(3) subunit) converted the resulting chimera to alphabetameATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non-alphabetameATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.

Adenosine Triphosphate↗

PubMed2001

Gene structure, chromosomal localization, cDNA cloning and expression of the mouse ATP-gated ionotropic receptor P2X5 subunit.

The P2X receptors are oligomeric ligand-gated ion channels operated by extracellular ATP. Here we report the genomic and cDNA sequence of the mouse P2X(5) subunit, as well as its genomic organization, chromosomal localization and expression in select tissues. The mouse gene spans approximately 13 kb of DNA and contains thirteen exons. The cDNA encodes a 455 amino acid protein with 95% identity to the rat subunit. The P2X(5) subunit gene encodes a 2.6 kb mRNA that was found to in a number of tissues, with highest levels detected in heart and kidney. Results from rapid amplification of cDNA ends (RACE) PCR suggest that there are multiple transcriptional start sites located approximately 30-70 bp upstream from the start codon. The gene was localized to band B5 on Chromosome 11 using fluorescence in-situ hybridization (FISH), a region that has a high degree of synteny with human Chromosome 17. These results provide the initial information useful for further investigation into the functional role(s) of the P2X(5) subunit in physiological processes.

Adenosine Triphosphate↗

PubMed2001

Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.

Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity ([Ca(2+)](0.5) of 25 and 83 microm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.

Calcium↗

PubMed2001

Quantitative analysis from CT is prognostic for local control of supraglottic carcinoma.

BACKGROUND: The purpose of this study was to determine whether pretreatment imaging with CT was prognostic for control of the primary site in patients with squamous cell carcinoma of the supraglottic larynx. METHODS: Pretreatment CT studies were obtained on 28 patients treated definitively with radiation therapy for supraglottic larynx cancer between 1991 and 1997. Follow-up ranged from 20-58 months. RESULTS: Local control was achieved in 61% of patients. Tumor volumes ranged from 0-68.6 cm(3), with a median of 3.1 cm(3). Local control rates for tumors with volumes greater than or less than 8 cm(3) were 20% and 70%, respectively (p =.0077). Mean tumor volumes for patients with and without recurrences were 10 cm(3) and 3.4 cm(3), respectively. CONCLUSIONS: This study demonstrates that quantitative analysis from CT imaging is prognostic for control of the primary site when radiation therapy is given for treatment of supraglottic cancer.

Carcinoma, Squamous Cell↗

PubMed2001

Treating benign colon disorders using laparoscopic colectomy.

Laparoscopic bowel surgery is a recent application of minimally invasive videoscopic techniques. Understanding the anatomy and physiology of the bowel, the background of bowel disorders and their treatment, signs and symptoms of bowel disease, and the patient selection process can help perioperative nurses better care for patients diagnosed with colon polyps, diverticulitis, and inflammatory bowel disease.

Colectomy↗

PubMed2001

Backbone dynamics of the regulatory domain of calcium vector protein, studied by (15)N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker.

UNLABELLED: CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. SUPPLEMENTAL MATERIAL: See www.proteinscience.org

Animals↗

PubMed2001

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves.

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

Amino Acid Sequence↗

PubMed2000

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily.

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.

Allosteric Regulation↗

PubMed2000

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.

Animals↗

PubMed2000

Electrocatalysis at a conducting composite electrode doped with a ruthenium(II) metallodendrimer.

A pentaerythritol-based metallodendrimer with RuIIterpyridine units was synthesized and tested as a mediator for the electrochemical oxidation of methionine (L-Met), cystine (L-Cys), and AsIII. A reversible oxidation of RuII was observed with the metallodendrimer as a solute in mixed acetonitrile-water solvents and as a component of carbon-based conducting composite electrodes. Mediated oxidation of the test species was observed. In aqueous solution, the composite electrode yielded a cyclic voltammetric peak current for the oxidation of L-Met in a 0.1 M phosphate buffer (pH 7.0) at 1.1 V vs Ag/AgCl. This anodic process was employed for amperometric detection in a flow system. Linear calibration curves were obtained over the range 1.0-10 microM Met and Cys. Using the criterion of the concentration yielding a signal 3 times the uncertainty of a blank, detection limits of 0.6 and 0.5 microM were calculated for Met and Cys, respectively. The slopes with three nominally identical electrodes varied by 10%.

2,2'-Dipyridyl↗

PubMed2000

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and functional characterization of the zebrafish ATP-gated ionotropic receptor P2X(3) subunit.

We describe a P2X subunit cloned from the zebrafish (Danio rerio) that is an orthologue of the mammalian P2X(3) subunit. Like the mammalian P2X(3), this receptor desensitizes rapidly in the presence of agonist. However, it differs in that alphabeta-meATP is a much less potent agonist than ATP and the antagonist TNP-ATP is not active at low nanomolar concentrations. Similar to the rat P2X(3) subunit, the zebrafish subunit forms hetero-oligomeric assemblies with the rat P2X(2) that possesses a phenotype distinct from either parent. This novel clone will provide an important basis for future experiments investigating the structure/function relationships of P2X subunit domains.

Amino Acid Sequence↗