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Biomedical subjects

B M Curtis

Publications and source records attributed to B M Curtis.

At least 19 recordsLinked to original sources
PubMed1992

Sequence and expression of a membrane-associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120.

The binding of the human immunodeficiency virus (HIV) envelope glycoprotein gp120 to the cell surface receptor CD4 has been considered a primary determinant of viral tropism. A number of cell types, however, can be infected by the virus, or bind gp120, in the absence of CD4 expression. Human placenta was identified as a tissue that binds gp120 in a CD4-independent manner. A placental cDNA library was screened by expression cloning and a cDNA (clone 11) encoding a gp120-binding protein unrelated to CD4 was isolated. The 1.3-kilobase cDNA predicts a protein of 404 amino acids with a calculated M(r) of 45,775 and organized into three domains: an N-terminal cytoplasmic and hydrophobic region, a set of seven complete and one incomplete tandem repeat, and a C-terminal domain with homology to C-type (calcium-dependent) lectins. A type II membrane orientation (N-terminal cytoplasmic) is predicted both by the cDNA sequence and by the reactivity of C-terminal peptide-specific antiserum with the surface of clone 11 transfected cells. Native and recombinant gp120 and whole virus bind transfected cells. gp120 binding is high affinity (kd, 1.3-1.6 nM) and inhibited by mannan, D-mannose, and L-fucose; once bound, gp120 is internalized rapidly. Collectively, these data demonstrate that the gp120-binding protein is a membrane-associated mannose-binding lectin. Proteins of this type may play an important role in the CD4-independent association of HIV with cells.

Amino Acid Sequence↗
PubMed1991

Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein.

Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast. Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains. The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone. This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.

Base Sequence↗
PubMed1991

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4.

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.

Amino Acid Sequence↗
PubMed1990

Expression cloning of a human granulocyte colony-stimulating factor receptor: a structural mosaic of hematopoietin receptor, immunoglobulin, and fibronectin domains.

We report the isolation from a placental library, of two cDNAs that can encode high affinity receptors for granulocyte colony-stimulating factor (G-CSF) when expressed in COS-7 cells. The cDNAs are predicted to encode integral membrane proteins of 759 and 812 amino acids in length. The predicted extracellular and membrane spanning sequences of the two clones are identical, as are the first 96 amino acids of their respective cytoplasmic regions. Different COOH termini of 34 or 87 residues are predicted for the two cDNAs, due apparently to alternate splicing. The receptor with the longer cytoplasmic domain is the closest human homologue of the murine G-CSF receptor recently described by Fukunaga et al. (Fukunaga, R., E. Ishizaka-Ikeda, Y. Seto, and S. Nagata. 1990. Cell. 61:341). A hybridization probe derived from the placental G-CSF receptor cDNA detects a approximately 3-kb transcript in RNAs isolated from placenta and a number of lymphoid and myeloid cells. The extracellular region of the G-CSF receptors is composed of four distinct types of structural domains, previously recognized in other cell surface proteins. In addition to the two domains of the HP receptor family-defining region (Patthy, L. 1990. Cell. 61:13) it incorporates one NH2-terminal Ig-like domain, and three additional repeats of fibronectin type III-like domains. The presence of both an NH2-terminal Ig-like domain and multiple membrane-proximal FN3-like domains suggests that the G-CSF receptor may be derived from an ancestral NCAM-like molecule and that the G-CSF receptor may function in some adhesion or recognition events at the cell surface in addition to the binding of G-CSF.

Amino Acid Sequence↗
PubMed1990

IL-1 and its receptor are translocated to the nucleus.

The internalization and intracellular transport of IL-1 and its receptor were examined in the murine T cell line EL-4. For 4 h after internalization intracellular 125I-IL-1 alpha remains bound to its receptor without degradation. Electron microscope autoradiography demonstrates that internalized IL-1 accumulates in purified nuclei. The IL-1 extracted from these nuclei is still bound to receptor. As no receptors for IL-1 were detected in untreated nuclei, these results suggest IL-1 driven translocation of the cell surface IL-1R complex to the nucleus. IL-1R internalization was correlated with IL-1 signal transduction events required to induce growth factor production from several subclones of EL-4 cells. The subsequent transport of the internalized IL-1R complex to the nucleus suggests the possibility for a nuclear site for IL-1R signaling.

Amino Acid Sequence↗
PubMed1989

T-cell interleukin 1 receptor cDNA expressed in Chinese hamster ovary cells regulates functional responses to interleukin 1.

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.

Animals↗
PubMed1987

Comparison of molecular properties of the voltage-sensitive Ca2+ channel from longitudinal smooth muscle of rabbit ileum with the channel purified from transverse tubules of rabbit skeletal muscle.

Dihydropyridine-sensitive calcium channels in membrane preparations from the longitudinal smooth muscle of rabbit ileum bound [3H]PN 200-110 with KD = 0.42 nM and Bmax = 265 fmol/mg. Calcium channels were labelled with [3H]PN 200-110 and solubilized with digitonin. The detergent-solubilized calcium channel had a sedimentation coefficient of 20.9 S in close agreement with the value of 20 S determined for the skeletal muscle calcium channel. Antibodies against the alpha-subunits of the skeletal muscle calcium channel specifically precipitated the calcium channel from smooth muscle. Our results suggest that the calcium channel from smooth muscle has a similar size and homologous alpha-subunits to those of the purified skeletal muscle calcium channel.

Animals↗
PubMed1986

Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules.

The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: alpha with Mr 135 000, beta with Mr 50 000, and gamma with Mr 33 000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular sieve chromatography. Forty-five percent of the alpha, beta, and gamma polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the alpha, beta, and gamma subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.

Animals↗
PubMed1985

Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by cAMP-dependent protein kinase.

Physiological studies indicate that voltage-sensitive calcium channels are regulated by cAMP and protein phosphorylation. The calcium antagonist receptor of the voltage-sensitive calcium channel from transverse-tubule membranes consists of three subunits, designated alpha, beta, and gamma. The catalytic subunit of cAMP-dependent protein kinase phosphorylates both the alpha and beta subunits of the purified receptor at a rate and extent that suggests they are potential physiological substrates of this enzyme. The phosphorylation of the alpha and beta subunits in transverse-tubule membranes was analyzed by two-dimensional gel electrophoresis. In intact transverse-tubule membranes, the alpha subunit is not significantly phosphorylated. However, the beta subunit, identified by its Mr, pI, and binding to wheat germ agglutinin-Sepharose, was one of the substrates selectively phosphorylated by cAMP-dependent protein kinase in transverse-tubule membranes. These results suggest that cAMP-dependent phosphorylation of the beta subunit of the calcium antagonist receptor may be an important regulatory mechanism for calcium channel function.

Animals↗
PubMed1984

Purification of the calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubules.

[3H]Nitrendipine binds with high affinity to a calcium antagonist receptor preferentially localized to the transverse tubule membrane of skeletal muscle. Digitonin was used to solubilize a [3H]nitrendipine-receptor complex from transverse tubule membranes. The digitonin-[3H]nitrendipine-receptor complex was purified 330-fold by a combination of wheat germ agglutinin chromatography, ion-exchange chromatography, and sedimentation through sucrose gradients to yield a preparation estimated to be 41% homogeneous on the basis of specific activity. Analysis by sodium dodecyl sulfate gel electrophoresis demonstrated that three polypeptides termed alpha [molecular weight (Mr) 130 000], beta (Mr 50 000), and gamma (Mr 33 000) quantitatively comigrated with the [3H]nitrendipine-receptor complex on sucrose gradients and represented 62% of the total protein staining. These three polypeptides are associated noncovalently. However, the apparent molecular weight of the alpha polypeptide is reduced from 160 000 to 130 000 upon reduction, consistent with the presence of an internal disulfide bond. Our results suggest that these three polypeptides are the subunits of the calcium antagonist receptor and are major components of the transverse tubule voltage-sensitive calcium channel.

Animals↗
PubMed1983

Solubilization of the calcium antagonist receptor from rat brain.

[3H]Nitrendipine binds with high affinity to a calcium antagonist receptor in rat brain membranes. At 4 degrees C, treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [3H]nitrendipine. The nitrendipine concentration that gave a half-maximal amount of the solubilized [3H]nitrendipine-receptor complex was identical to the Kd for specific nitrendipine binding to brain membranes. Nitrendipine dissociated from digitonin-solubilized and membrane-bound receptors with a half-time of 24 to 30 min at 20 degrees C. Verapamil increased and diltiazem decreased the dissociation rate to a similar extent in both preparations indicating that the solubilized receptor contains both the dihydropyridine and diltiazem/verapamil binding sites. Sucrose gradient sedimentation experiments gave a value of S20, omega = 19.2 for the receptor-digitonin complex. The solubilized calcium antagonist receptor binds specifically to wheat germ agglutinin-Sepharose columns consistent with an identification as a glycoprotein.

Animals↗
PubMed1983

Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat.

We have developed a method for labeling retinal ganglion cells in neonatal rats by retrograde transport of the fluorescent dye, True Blue (TB), injected into the optic chiasm. Following proteolytic dissociation of labeled retinas into single cells, the labeled cells could be enriched 50- to 100-fold by centrifugation in a 5%/10% metrizamide gradient. When plated in Ham's F-10 medium in the presence of fetal calf serum and chick optic tectum-conditioned medium, the labeled cells could be maintained in vitro up to 48 hr. In these cultures, the ganglion cells (GCS) constituted 50 to 70% of the total cell population. When GC-rich fractions or GC cultures were stained with a monoclonal antibody to Thy-1 antigen, greater than 90% of the TB-labeled cells were reactive. In order to localize voltage-sensitive sodium channels, GC-rich cultures were reacted with 125I-scorpion toxin. Analysis of the autoradiograms showed that the density of silver grains was about 10-fold higher on TB-labeled cells than on nonfluorescent cells, or in controls which contained excess of unlabeled toxin. When GC cultures were incubated with micromolar concentrations of putative GC transmitters, aspartate and glutamate, the amino acids were accumulated by 15 to 20% of labeled cells. Several lectin receptors were also localized on TB-labeled cells in situ. Whereas the lectins wheat germ agglutinin, concanavalin A, peanut agglutinin, Dolichos biflorus agglutinin, and Limulus polyphemus agglutinin bound to TB-labeled cells, others such as Ricinus communis agglutinin I, Ulex, and Lotus lectins showed no binding. The lectin binding was specific since preincubation with the appropriate hapten sugar blocked lectin binding.

Animals↗
PubMed1981

The mechanism of action of local anesthesia by tetraethylammonium derivatives.

Tetraethylammonium (TEA+) derivatives in which one of the ethyl groups is replaced by a longer chain alkane (C4-C16) were tested for their ability to block the compound action potential (CAP) of frog sciatic nerve. A parabolic relation between chain length and 100 per cent blocking dose was found, suggesting an optimal size of C12 for the inhibitory receptor. Two types of inhibition were observed. Type 1 was an inhibition of the CAP at all frequencies when the nerve was perfused with the TEA+ derivative. Interaction with tetrodotoxin suggests this is a Na+ channel effect. Type 2 was an irreversible (still present after nerve wash) frequency-dependent inhibition that is distinct from and synergistic with Na+ channel blocking local anesthetics. From the kinetics of inhibition onset and transport studies, it is suggested that the ultralong action of the TEA+ derivatives is mediated by binding to a receptor at the internal part of K+ channels.

Anesthetics, Local↗
PubMed1981

Tetraethylammonium derivatives: ultralong-acting local anesthetics?

Derivatives of tetraethylammonium ion (TEA+) were synthesized in which one ethyl group was replaced by a C6, C8, C10, C12, C14, or C16 side chain. These TEA+ derivatives were tested for duration of sensory block of the rat infraorbital (trigeminal) nerve. The duration of sensory anesthesia increased exponentially from 1.2 hours to 388 hours as chain length increased from C2-C12, while C12, C14, and C16 all produced a similar reversible block of 17--20 days. The block duration of C12 was correlated with C12 bound to the infraorbital nerve; C12 not bound by the nerve was quantitatively excreted by the kidneys. These data, along with the lack of observable microscopic toxicity, suggests that TEA+ derivatives may be a useful new class of ultralong-acting local anesthetics.

Anesthetics, Local↗