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Results for “Functional characterization”

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PubMed1996

Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H-NMR.

Cooperativity between redox and protonation centres is known to be crucial for the function of complex proteins, but it is often difficult to describe in terms of thermodynamic parameters. Cytochrome c3 is a good model for these studies since, while retaining the overall complexity of larger systems, it is suitable for detailed crystallographic and spectroscopic studies. Assignment of the haem substituent NMR resonances, together with NMR redox titrations of cytochrome c3 from D. desulfuricans ATCC 27774, was used to correlate relative redox potentials to specific haems in the structure: haem II approximately equal to haem I < haem IV < haem III. This order is different from that determined for the homologous proteins studied and in disagreement with that previously reported for this cytochrome (Morais, J., Palma, N., Frazäo, C., Caldeira, J., LeGall, J., Moura, I., Moura, J.J.G. and Carrondo, M.A. (1995) Biochemistry 34, 12830-12841).

Cytochrome c Group↗

PubMed2004

Functional characterization of brain peptide transporter in rat cerebral cortex: identification of the high-affinity type H+/peptide transporter PEPT2.

In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P(2) fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [14C]glycylsarcosine (Gly-Sar), a substrate for H(+)/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degrees C by a rapid filtration technique. The uptake of [14C]Gly-Sar into synaptosomes was stimulated by an inwardly directed H(+)-gradient. The uptake system exhibited a Michaelis-Menten constant (K(t)) of 110+/-20 microM for Gly-Sar. This value is comparable to the K(t) value for Gly-Sar uptake via the high-affinity H(+)/peptide transporter PEPT2. The H(+)-dependent uptake of [14C]Gly-Sar into synaptosomes was inhibited by di- and tripeptides and beta-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr-Arg) completely inhibited Gly-Sar uptake with the K(i) value of 29+/-14 microM. These uptake properties of the brain peptide transporter (i.e., the K(t) value for Gly-Sar uptake and the K(i) value of kyotorphin for Gly-Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H(+)-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2.

Animals↗

PubMed2006

Functional characterization of parvalbumin from the Arctic cod (Boreogadus saida): similarity in calcium affinity among parvalbumins from polar teleosts.

Calcium dissociation constants (KD) were measured as a function of temperature for parvalbumin, a small acidic protein expressed abundantly in fast-twitch muscle, from the Arctic cod (Boreogadus saida) and compared to values previously determined for Antarctic and temperate zone teleosts. Estimates of KD were derived independently from fluorometric titrations and calorimetry. In addition, the primary structure of B. saida parvalbumin was determined. Calcium KDs for parvalbumin from B. saida were fundamentally similar to those for parvalbumins from Antarctic species (6.68+/-0.59 nM and 7.77+/-0.72 nM at 5 degrees C, respectively), but significantly different from temperate zone species (1.35+/-0.28 nM at 5 degrees C). However, estimates of KD for B. saida parvalbumin at 5 degrees C closely matched values for temperate zone fish at 25 degrees C (6.54+/-0.56 nM), recapitulating the prior observation that calcium affinity of parvalbumin is conserved at the native temperature of teleost fish. Full sequence of B. saida parvalbumin was generated using reverse-phase HPLC and RACE-PCR. The Arctic parvalbumin showed 83% homology to a carp parvalbumin. None of the 16 total substitutions between the two parvalbumins resided in the cation binding sites of the protein, indicating that the structural locus of the thermal sensitivity of function lies outside the active regions.

Amino Acid Sequence↗

PubMed2005

The putative protein 6 of the severe acute respiratory syndrome-associated coronavirus: expression and functional characterization.

The SARS-CoV open reading frame 6 (ORF6) is transcribed into mRNA6 and encodes a putative 7.5 kDa accessory protein, SARS 6, with unknown function. In this study, we have confirmed the SARS 6 protein expression in lung and intestine tissues of the SARS patients and in SARS-CoV infected Vero E6 cells by immunohistochemistry. Further studies by immunoblot and confocal microscopy analyses revealed the expression and the endoplasmic reticulum (ER) localization of the recombinant SARS 6 protein in mammalian cells. Expression of SARS 6 protein in mammalian cells elicits biological activity of stimulating cellular DNA synthesis.

Amino Acid Sequence↗

PubMed2005

Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.

Amino Acid Sequence↗

PubMed2000

Molecular identification and functional characterization of Mdr1a in rat cholangiocytes.

BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure. We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes. METHODS: A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers. Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively. Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918. RESULTS: A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum. Western analysis localized mdr1 to the apical membrane of cholangiocytes. Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner. CONCLUSIONS: These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Molecular and functional characterization of gap junctions in the avian inner ear.

To analyze the fundamental role of gap junctions in the vertebrate inner ear, we examined molecular and functional characteristics of gap junctional communication (GJC) in the auditory and vestibular system of the chicken. By screening inner ear tissues for connexin isoforms using degenerate reverse transcription-PCR, we identified, in addition to chicken Cx43 (cCx43) and the inner-ear-specific cCx30, an as yet uncharacterized connexin predicted to be the ortholog of the mammalian Cx26. In situ hybridization indicated that cCx30 and cCx26 transcripts were both widely expressed in the cochlear duct and utricle in an overlapping pattern, suggesting coexpression of these isoforms similar to that in the mammalian inner ear. Immunohistochemistry demonstrated that cCx43 was present in gap junctions connecting supporting cells of the basilar papilla, in which its immunofluorescence colocalized with that of cCx30. However, cCx43 was absent from supporting cell gap junctions of the utricular macula. This variation in the molecular composition of gap junction plaques coincided with differences in the functional properties of GJC between the auditory and vestibular sensory epithelia. Fluorescence recovery after photobleaching, adapted to examine the diffusion of calcein in inner ear explants, revealed asymmetric communication pathways among supporting cells in the basilar papilla but not in the utricular macula. This study supports the hypothesis that the coexpression of Cx26/Cx30 is unique to gap junctions in the vertebrate inner ear. Furthermore, it demonstrates asymmetric GJC within the supporting cell population of the auditory sensory epithelium, which might mediate potassium cycling and/or intercellular signaling.

Animals↗

PubMed1993

Structural and functional characterization of the HPV16 E7 protein expressed in bacteria.

The E7 gene of the human papillomaviruses (HPV) encodes a 98-amino acid, multifunctional nuclear phosphoprotein with functional and structural similarities to adenovirus E1A and the papovavirus T antigens. E7 is a viral oncoprotein, which will cooperate with an activated ras oncogene to transform primary rodent cells, and can cooperate with the HPV E6 protein for the efficient immortalization of primary human keratinocytes. Due to the compelling epidemiological and experimental association between HPV infection and cervical cancer, we have undertaken a detailed study of the structure of the HPV16 E7 protein. The E7 protein was expressed in Escherichia coli as a native, unfused polypeptide, and soluble protein was purified by conventional chromatographic techniques. The purified protein was assessed for various biochemical and biophysical properties. Purified E7 binds the retinoblastoma protein avidly and specifically, and it can dissociate the E2F transcription factor when assayed in vitro. Circular dichroism spectroscopy indicated that E7 reversibly binds Zn2+ and Cd2+, resulting in a substantial increase in the alpha-helical content of the metal-bound E7 consistent with the stabilization of a hydrophobic core in the COOH terminus of the protein.

Amino Acid Sequence↗

PubMed1984

Structural and functional characterization of the RNA-positive complementation groups, C and D, of the New Jersey serotype of vesicular stomatitis virus: assignment of the M gene to the C complementation group.

The structural and functional lesions in the RNA-positive complementation groups, C and D, of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus have been characterized. The M protein of the temperature-sensitive mutant C1, the prototype of the C complementation group, was degraded at the restrictive temperature in vivo, and was resolved from the wild-type M protein by SDS-polyacrylamide gel electrophoresis and nonequilibrium pH gradient electrophoresis. Coreversion of these properties and the temperature-sensitive phenotype was observed in a spontaneous revertant. On the basis of these results, the M gene was assigned to the C complementation group. Intracellular nucleocapsids could not be isolated from New Jersey serotype infections by procedures developed for Indiana serotype infections. Therefore, in order to assess the ability of New Jersey ts mutants to accumulate nucleocapsids at the restrictive temperature, a procedure for their isolation was developed. Hypertranscription was observed in C1-infected cells incubated at the restrictive temperature, but was not accompanied by proportionate increases in intracellular viral nucleocapsids or protein synthesis. The G and N proteins of the temperature-sensitive mutant D1, the sole representative of the D complementation group, were electrophoretic variants. The relative yield of intracellular D1 N protein was lower at the restrictive than at the permissive temperature, and the D1 L protein was thermolabile. No intracellular viral nucleocapsids were detected in D1 infected cells incubated at the restrictive temperature; however, more 40 S and less message-sized RNA were synthesized at the restrictive than at the permissive temperature. These results suggested functional defects in both the N protein and polymerase of D1.

Animals↗

PubMed2026

Structural and functional characterization of peanut expansin proteins identifies AhEXPA3 as a stress-responsive regulator of seed germination.

Expansins are cell wall-associated proteins that play important roles in plant growth, development, and environmental responses, yet their structural features and functional significance in peanut remain insufficiently understood. Here, we performed a genome-wide identification and characterization of 70 expansin proteins in cultivated peanut. Phylogenetic analysis classified these genes into four subfamilies (EXPA, EXPB, EXLA, and EXLB), with conserved motif patterns and subgroup-specific exon-intron structures. Collinearity and evolutionary analyses revealed that segmental duplication mainly drove peanut expansin family expansion, with most duplicated gene pairs subsequently undergoing purifying selection. Promoter analysis identified abundant cis-regulatory elements associated with light responses, hormone signaling, and stress responses. Expression profiling indicated that many AhEXP genes were differentially expressed during seed germination and in response to abscisic acid (ABA), salt, and osmotic stresses. Among them, AhEXPA3 was identified as a stress-responsive expansin protein with marked transcriptional induction under abiotic stress conditions. Subcellular localization analysis suggested that AhEXPA3 exhibits an endoplasmic reticulum-associated localization pattern. Functional assays further demonstrated that heterologous expression of AhEXPA3 inhibited seed germination and early seedling establishment under stress conditions in both Arabidopsis thaliana and rice. These findings support a negative regulatory role of AhEXPA3 in stress-responsive seed germination and broaden current understanding of expansin protein function in legumes.

Germination↗

PubMed2004

Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system.

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.

Amacrine Cells↗

PubMed2000

Molecular cloning and functional characterization of murine transmembrane activator and CAML interactor (TACI) with chromosomal localization in human and mouse.

The human Taci gene (Transmembrane Activator and CAML Interactor) encodes a recently discovered member of the Tumor Necrosis Factor Receptor family. TACI is expressed in B-lymphocytes and may act to regulate humoral immunity. To identify functionally important regions of the protein, we have isolated and characterized the murine homolog of the human Taci cDNA. The proteins display 61.5% similarity and 54.6% identity. Mouse TACI is a type III transmembrane protein, as judged by the lack of a cleaved signal sequence and its N-terminal extracellular exposure. The intracellular domains of the mouse and human proteins share a single, defined region of high sequence conservation (19 of 23 residues identical). This constitutes a novel domain that may play a part in the initiation of signal transduction through TACI. In support of this notion, mouse TACI was found to activate NFAT, NFkB, and AP1 transcription factors in a transient transfection assay. The Taci gene was localized to human Chromosome (Chr) 17p11 by fluorescence in situ hybridization. The murine homolog was localized by intraspecific backcross analysis to the middle of Chr 11, a region that is syntenic to human Chr 17p. This work identifies conserved domains within TACI that may mediate the cellular distribution and signal transduction function of the protein and extend the details of homology between mouse Chr 11 and human 17p.

Amino Acid Sequence↗

PubMed2001

Human cystinuria-related transporter: localization and functional characterization.

BACKGROUND: Cystinuria has been proposed to be an inherited defect of apical membrane transport systems for cystine and basic amino acids in renal proximal tubules. Although the mutations of the recently identified transporter BAT1/b(0,+)AT have been related to nontype I cystinuria, the function and localization of human BAT1 (hBAT1)/b(0,+)AT have not been well characterized. METHODS: The cDNA encoding hBAT1 was isolated from human kidney. Fluorescence in situ hybridization was performed to map the hBAT1 gene on human chromosomes. Tissue distribution and localization of expression were examined by Northern blot and immunohistochemical analyses. hBAT1 cDNA was transfected to COS-7 cells with rBAT cDNA, and the uptake and efflux of 14C-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibitors or activators of protein kinases. RESULTS: The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, which is the locus of nontype I cystinuria. hBAT1 message was expressed predominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0,+)-amino acid transporter), hBAT1 exhibited the transport activity with the properties of amino acid transport system b(0,+), which transported cystine as well as basic and neutral amino acids presumably via a substrate exchange mechanism. BAT1-mediated transport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. CONCLUSIONS: hBAT1 exhibited the properties expected for a transporter subserving the high-affinity cystine transport system in renal proximal tubules. The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirming the involvement of hBAT1 in cystinuria.

ATP-Binding Cassette Transporters↗

PubMed1998

Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa).

Recently, a polyspecific organic cation transporter, hOCT1, was cloned from human liver. To date, limited studies examining the functional characteristics of the transporter have been performed. The purpose of the present study was to develop a mammalian expression system for hOCT1 and to characterize the interactions of various compounds with the cloned transporter. Lipofection was used to transiently transfect the hOCT1 plasmid DNA in a human cell line, HeLa. We tested the interaction of an array of organic cations and other compounds with hOCT1 by determining Ki values in inhibiting 14C-tetraethylammonium (TEA) transport in the transfected cells. Transient expression of hOCT1 activity was observed between 24 and 72 hr post-transfection, with maximal expression at approximately 40 hr. TEA transport was temperature dependent and saturable with Vmax and K(m) values of 2.89 +/- 0.448 nmol/mg protein/30 min and 229 +/- 78.4 microM, respectively. 14C-TEA uptake in hOCT1 plasmid DNA-transfected HeLa cells was trans-stimulated by unlabeled TEA and 1-methyl-4-phenyl-pyridinium. Organic cations, including clonidine, quinine, quinidine and verapamil (0.1 mM), significantly inhibited 14C-TEA uptake, whereas the organic anion, p-aminohippuric acid (5 mM), did not. The neutral compounds, corticosterone (Ki, 7.0 microM) and midazolam (Ki, 3.7 microM) potently inhibited 14C-TEA uptake. The Ki values of several compounds in interacting with hOCT1 differed substantially from the corresponding values for the rat organic cation transporter, rOCT1, and the human kidney-specific organic cation transporter, hOCT2, determined in previous studies. Transiently transfected HeLa cells represent a useful tool in studying the interactions and kinetics of organic cations and other xenobiotics with hOCT1 and in understanding the molecular events involved in organic cation transport.

Carrier Proteins↗

PubMed2003

Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: destructive potential in vitro and in vivo.

OBJECTIVE: To characterize the morphologic and immunologic features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and to explore their potential function in vitro and in vivo by focusing on cartilage destruction. METHODS: Synovial fluid adherent cells obtained from patients with RA and from control subjects were characterized by immunohistochemistry, flow cytometry, and electron microscopy. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulfated glycosaminoglycans (sGAG) into the medium, and the level of matrix metalloproteinase 1 (MMP-1) in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. To inhibit cartilage destruction in vitro, the MMP inhibitor marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule and maintained there for 60 days. RESULTS: In vitro, the synovial fluid adherent cells consisted of 2 subpopulations, large round-shaped macrophage-like cells (CD68+) and spindle-shaped fibroblast-like cells (Thy-1+). When passaged, the latter cells proliferated and organized themselves into 3-dimensional formations. This allowed them to reach collagen particles fixed with agarose. Fibroblasts derived from synovial tissues could not be used in this assay because they grew only in monolayers and not on agarose. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy-1+,CD45-,CD68-,CD86- phenotype. Electron microscopy did not reveal important morphologic differences between the 2 types of fibroblasts, those from synovial tissue or those from synovial fluid. However, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin 3, as well as the complement-regulatory molecule CD55. The in vitro release of sGAG associated with cell activity was 2.5-fold higher from RA synovial fluid adherent cells in comparison with that from negative control cells. The release of sGAG correlated with the concentration of MMP-1 and was inhibited by the broad-range MMP inhibitor marimastat in a dose-dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behavior as that displayed by tissue-derived RA synovial fibroblasts. CONCLUSION: Similar to tissue-derived RA synovial fibroblasts, RA synovial fluid adherent cells, which contain "floating" anchorage-independent fibroblast-like cells, mediate cartilage destruction independent of the hyperplastic synovial tissue.

Adult↗

PubMed1986

Functional characterization of toposomes from sea urchin blastula embryos by a morphogenetic cell aggregation assay.

This paper documents the evidence that the large oligomeric glycoprotein complexes of unknown function first isolated as 22S particles from sea urchin embryos are the sole agents responsible for the adhesive integrity of sea urchin blastula embryos. The conclusion rests on the demonstration that polyclonal IgG (as serum or monovalent Fab) against whole membranes or butanol-solubilized components of membranes, as well as against the purified particle itself, completely blocks reaggregation of dissociated blastula cells and that this inhibition is reversed by neutralization of the inhibitory antibodies with purified 22S antigen. An essential aspect of the evidence is the combination of quantitative endpoint titrations in microtiter wells with the qualitative parameters of morphogenesis. The new data complement previous evidence that morphogenesis is mediated by a general class of particles, toposomes, responsible for mechanical linkage between cells and their positional guidance in embryogenesis.

Animals↗

PubMed1999

Functional characterization of alpha1-adrenergic receptors in experimental vein grafts.

BACKGROUND: Studies on the pharmacology of the smooth muscle cells in vein bypass grafts suggest that the function of G-proteins and adrenergic receptors is altered. This study examines the alpha-adrenergic responsiveness of smooth muscle cells in vein bypass grafts as compared with those in the common carotid arteries and external jugular veins. METHODS: New Zealand White rabbits received jugular vein interposition bypass grafts of the common carotid. Vessel segments of the vein bypass grafts harvested after 28 days, common carotid arteries, and external jugular veins were sectioned into 5-mm rings (four per vessel) for studies of isometric tension in response to phenylephrine (10(-10) to 10(-4) M) alone and in the presence of prazosin, an alpha1-adrenergic antagonist; WB4101 and 5-methylurapidil (5-MU), alpha1A antagonists; chloroethylclonidine (CEC); an alpha1B antagonist; or the Gi/o G-protein inhibitor pertussis toxin (PTx). RESULTS: All vessels had prazosin-sensitive responses. The jugular veins appear to have functional alpha1A receptors (WB4101 and 5-MU sensitive, CEC insensitive) which are associated with pertussis toxin-sensitive G-proteins. Carotid arteries appear to have atypical alpha1 receptors (WB4101 and 5-MU insensitive, CEC insensitive) associated with pertussis toxin-insensitive G-proteins. Vein grafts appear to have functional alpha1B receptors (WB4101 and 5-MU insensitive, CEC sensitive) which are associated with pertussis toxin-insensitive G-proteins. CONCLUSIONS: These results show that placement of a vein into the arterial circulation induces a change in alpha1-adrenergic receptor subtypes (alpha1A to alpha1B) and in the G-protein coupling of the receptors (PTx sensitive to PTx insensitive), reflecting a signficant phenotypic change in smooth muscle cell signal transduction.

Adrenergic alpha-Agonists↗

PubMed1985

Topographical analysis of bovine herpesvirus type-1 glycoproteins: use of monoclonal antibodies to identify and characterize functional epitopes.

Monoclonal antibodies, specific for two of the major bovine herpesvirus type-1 (BHV-1) glycoproteins, GVP 3/9 and GVP 6/11a/16, have been previously produced and characterized. The reactivity of each monoclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA), the ability to neutralize viral infectivity, and the capacity to mediate complement-dependent (AbC') lysis of virus-infected cells. The topography of epitopes on GVP 3/9 and GVP 6/11a/16 was analyzed, using selected monoclonal antibodies in a competitive antibody binding assay (CBA). Nine epitopes were identified on GVP 3/9. Comparison of the biological activities and epitope specificities of the monoclonal antibodies against GVP 3/9 showed that one epitope was involved in virus neutralization, whereas six epitopes mediated AbC' lysis of the virus-infected cell. Of the seven epitopes identified on GVP 6/11a/16, six were involved in neutralization and three participated in AbC' lysis. In some cases, partial competition between monoclonal antibodies was observed, indicating that they were directed against overlapping or closely adjacent epitopes. In other instances, asymmetrical competition between monoclonal antibodies suggested conformational changes in the glycoprotein molecule induced by antibody binding. Mixtures of two monoclonal antibodies with different epitope specificities resulted in higher neutralizing activity than either antibody alone, suggesting that multiple monoclonal antibodies can exert a synergistic effect on virus neutralization.

Animals↗