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Biomedical subjects

C D Richardson

Publications and source records attributed to C D Richardson.

At least 37 records · Page 2Linked to original sources
PubMed1997

In situ hybridization: identification of rare mRNAs in human tissues.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Humans↗
PubMed1997

Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding.

CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus.

A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory. This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies. The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced. The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element. A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified. Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection. We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus. The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE).

Interferon regulatory factor (IRF) genes encode a family of DNA-binding proteins that are involved in the transcriptional regulation of type-I interferon and/or interferon-inducible genes. We report here the characterization of LSIRF, a new member of the IRF gene family cloned from mouse spleen by the polymerase chain reaction using degenerate primers. LSIRF was found to encode a 51 kDa protein that shares a high degree of amino acid sequence homology in the DNA-binding domain with other IRF family members. LSIRF expression was detectable only in lymphoid cells. In contrast to other IRF genes, LSIRF expression was not induced by interferons, but rather by antigen-receptor mediated stimuli such as plant lectins, CD3 or IgM crosslinking. In in vitro DNA binding studies, LSIRF was able to bind to the interferon-stimulated response element (ISRE) of the MHC class I promoter. The expression pattern and DNA binding activities suggest that LSIRF plays a role in ISRE-targeted signal transduction mechanisms specific to lymphoid cells.

Amino Acid Sequence↗
PubMed1994

CD46, a primate-specific receptor for measles virus.

Measles virus normally infects only primate cells. The receptor for measles virus has recently been shown to be the complement regulator CD46, also known as membrane cofactor protein. Transfection of rodent cells with human CD46 renders them susceptible to the virus, suggesting that transgenic animals may prove useful for testing antiviral agents and vaccines.

Amino Acid Sequence↗
PubMed1993

The human CD46 molecule is a receptor for measles virus (Edmonston strain).

Measles virus normally causes disease in human beings, and the host range of this virus may be determined by a specific receptor on the surface of primate cells. Human-rodent somatic cell hybrids were tested for their ability to bind measles virus, and only cells that contained human chromosome 1 were capable of binding virus. A study of lymphocyte markers suggested that the complement regulator known either as membrane cofactor protein or CD46 was the measles virus receptor. We proved this hypothesis by demonstrating that hamster cell lines that expressed human CD46 could subsequently bind virus. Furthermore, infected CD46+ cells produced syncytia and viral proteins. Finally, polyclonal antisera against CD46 inhibited virus binding and infection. These results prove that human CD46 permits cells both to bind measles virus and to support infection.

Animals↗
PubMed1993

Structural requirements for the inhibition of membrane fusion by carbobenzoxy-D-Phe-Phe-Gly.

The peptide ZfFG is known to inhibit non-bilayer phase formation as well as vesicle-vesicle and viral fusion. In order to ascertain some of the properties or structural features of this peptide which were important for the inhibition of membrane fusion, the blocking group was transferred from the amino to the carboxyl end to make fFGOBz. The fFGOBz lowered the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine and it promoted the formation of isotropic phases in monomethyldioleoylphosphatidylethanolamine. The promotion of non-bilayer phases by fFGOBz appeared to be enhanced by a charged terminal amino group as higher pH or formylation of the amino group both decreased the effectiveness of this peptide to induce formation of the hexagonal phase. With the monomethyldioleoylphosphatidylethanolamine, the fFGOBz also promoted vesicle leakage and fusion as measured by lipid intermixing. The fFGOBz did not inhibit the formation of lipid structures of high curvature, resulting from sonication of phosphatidylcholine, as did ZfFG. Thus, the effects of fFGOBz on membranes are in sharp contrast to those of ZfFG and more closely resemble the behaviour of larger fusion peptides corresponding to the amino-terminal segment of viral fusion proteins. Our results demonstrate that having the carbobenzoxy group on the amino-terminus of fFG is important for giving the peptide derivative the property of inhibiting membrane fusion.

Amino Acid Sequence↗
PubMed1993

5-lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase.

5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA4 from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-LO, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA4. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H-indol-2-yl]-2,2- dimethyl-propanoic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein.

5-Lipoxygenase-Activating Proteins↗
PubMed1993

The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.

Animals↗
PubMed1992

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.

Amino Acid Sequence↗
PubMed1992

Sequence analysis of human coronavirus 229E mRNAs 4 and 5: evidence for polymorphism and homology with myelin basic protein.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.

Amino Acid Sequence↗
PubMed1992

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.

Amino Acid Sequence↗
PubMed1992

Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.

Animals↗
PubMed1991

Effects of the 'fusion peptide' from measles virus on the structure of N-methyl dioleoylphosphatidylethanolamine membranes and their fusion with Sendai virus.

31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of the membrane protein gene of human coronavirus 229E.

Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of a baculovirus occlusion body glycoprotein which resembles spheroidin, an entomopoxvirus protein.

A 37-kDa polypeptide specified by Autographa californica nuclear polyhedrosis virus was found to share significant homology with Choristoneura biennis entomopoxyvirus spheroidin protein, which is the major component of entomopoxvirus occlusion bodies. Antibodies raised against spheroidin cross-reacted with the 37-kDa protein and confirmed its expression in the late phase of wild-type baculovirus infection. Immunoblot analysis and fluorescence microscopy demonstrated that the protein was associated with purified A. californica nuclear polyhedrosis virus occlusion bodies and was absent in purified virions. Immunofluorescence studies localized the protein to the periphery of occlusion bodies and the internal membranes of cells infected with wild-type baculovirus. The open reading frame encoding this spheroidinlike protein was inserted into a baculovirus expression vector, and recombinant protein was synthesized under control of the polyhedrin promoter. Studies of the recombinant protein demonstrated that it was heterogeneous in molecular mass as a result of N-linked glycosylation. Tunicamycin inhibited carbohydrate addition and yielded proteins of 34 and 33 kDa.

Amino Acid Sequence↗