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Biomedical subjects

M D Parker

Publications and source records attributed to M D Parker.

At least 19 recordsLinked to original sources
PubMed2006

Ischemic epigenetics and the transplanted kidney.

The primary purpose of this investigation was to study oxidative demethylation of DNA following ischemia/reperfusion injury (I/RI) that putatively influences posttransplant gene expression in transplanted kidneys. Our hypothesis was that as a result of I/RI, oxidative damage, which is inherent in solid organ transplantation, may lead to aberrant demethylation of cytosine-guanine (CpG) sites within gene promoter regions of DNA. The methylated CpG sites normally contribute to the binding of proteins that render DNA inaccessible to transcription factors. Therefore, conversion of methylated cytosines to nonmethylated cytosines by oxidative damage in postischemic organs might facilitate enhanced gene expression in donor organs by exposing the demethylated CpG site in a gene promoter to DNA-binding proteins that enhance gene transcription. In this study, we investigated the demethylation of a specific CpG within the IFNgamma response element resident in the promoter region of the C3 gene in the rat kidney. In response to 24 hours of cold ischemia and a subsequent 2 hours of reperfusion in an isolated ex-vivo circuit, we observed a significant change in the ratio of methylated to unmethylated cytosines at this site. Epigenetic modifications to donor DNA have not been previously investigated, but our own data suggests that they have the potential to modify gene expression posttransplantation. Since epigenetic modification may become stable and heritable upon mitosis, such changes to the donor organ DNA may persist with enormous implications for transplant outcomes.

Animals↗
PubMed2001

Human BTR1, a new bicarbonate transporter superfamily member and human AE4 from kidney.

We report the cloning, characterization, and chromosomal localization of two novel human members of the bicarbonate transporter superfamily, BTR1 (Bicarbonate Transporter Related protein-1) and AE4 (Anion Exchange protein 4). BTR1 is a novel mammalian protein. The BTR1 gene maps to chromosome 20p12 and encodes a 100 kDa protein predominantly expressed in the kidney, salivary glands, testis, thyroid glands, and trachea. The AE4 gene maps to chromosome 5q23-31 and encodes a 104 kDa protein expressed mainly in the kidney. Human AE4 shares 84% identity with the recently reported rabbit AE4, a sodium independent, Cl(-)/HCO(-)(3) exchanger located on the apical membrane of beta-intercalated kidney cells.

Amino Acid Motifs↗
PubMed2001

Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

Animals↗
PubMed2000

Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Adenine↗
PubMed2000

Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.

The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucleotide differences in 8624 bases (1.7% divergence), of which only 28% (42 nucleotides) altered the encoded amino acids. Comparison of deduced nsP1 and nsP4 amino acid sequences from WEE with the corresponding proteins from eastern equine encephalitis virus (EEE) yielded identities of 84.9 and 83.8%, respectively. Previously uncharacterized stem-loop structures were identified in the nontranslated terminal regions. A cDNA clone of the 26S region encoding the structural polyprotein of WEE strain 71V-1658 was placed under the control of a cytomegalovirus promoter and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies that recognize WEE antigens, thus, forming the initial step in the investigation of subunit vaccines to WEE.

5' Untranslated Regions↗
PubMed1996

Complete sequence of Venezuelan equine encephalitis virus subtype IE reveals conserved and hypervariable domains within the C terminus of nsP3.

The complete nuleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97--100% identity) and more distantly related to VEE strains of other antigenic varieties (83--93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94--95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1--329), which is highly conserved among all alphaviruses, was 93--94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330--563) was only 59--61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts.

Amino Acid Sequence↗
PubMed1996

Urothelial striations revisited.

PURPOSE: To confirm that use of low-osmolality contrast media (LOCM) in urography increases the frequency with which striations occur in otherwise healthy adults. MATERIALS AND METHODS: Two series of urograms obtained in 207 adult patients (101 men, 106 women) at two institutions after the administration of either LOCM or high-osmolality contrast media (HOCM) were reviewed. At the first, 106 patients had received LOCM; at the second, 101 had received HOCM. Protocols were similar. Urograms were evaluated for the presence and extent of striations. RESULTS: Of 106 LOCM urograms, 16 (15%) showed striations. Of 101 HOCM urograms, only two (2%) showed striations. The difference in striations shown between the two groups was significant (P < .002). Striations were most common in renal pelves and distal ureters and on radiographs obtained after voiding. CONCLUSION: Detection of urothelial striations on LOCM urograms in adults is most often normal. Increased prevalence of striations is likely due to decreased urinary tract distention associated with LOCM use.

Adult↗
PubMed1995

Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus.

We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.

Animals↗
PubMed1994

Respiratory abnormalities in infants of substance-abusing mothers: role of prematurity.

It is a current hypothesis that maternal history of drug addiction during pregnancy and detection of drugs in the urine of the newborn are associated with increased incidence of apnea. To test this hypothesis, we reviewed polygraphic studies of respiration in two groups of infants who had been exposed in utero to cocaine (and other drugs). The first group was composed of 20 term infants (39.1 +/- 0.8 weeks gestation), and premature infants (35.4 +/- 0.8 weeks gestation). None of the infants were on methylxanthines. These infants were matched with 15 term and 15 preterm infants of similar gestational age. Variables studied were: heart rate, respiration, chest impedance pneumography, nasal airflow and oxygen saturation (pulse oximetry). Apnea indices for central and obstructive events of short and long duration as well as periodic breathing and oxygen saturation were obtained. Term drug-exposed infants had less central apnea and a higher rate of periodic breathing compared to term controls, whereas drug-exposed premature infants had more obstructive apnea and less periodic breathing compared to premature controls. These observed differences within groups were subtle and clinically insignificant. Other parameters studied were not different. When term and preterm infants were compared, preterm infants had significantly higher central apnea, obstructive apnea and periodic breathing rates. These differences appeared to be related to gestational age differences, not to drug exposure. There was no evidence that exposure to cocaine and other drugs actually inhibited respiration.

Adult↗
PubMed1993

Assembly of recombinant rotavirus proteins into virus-like particles and assessment of vaccine potential.

Rotavirus structural proteins VP4, VP6 and VP7 from Bovine Rotavirus Strain C486 were cloned and expressed in a baculovirus expression system. Combinations of the proteins were assembled into a series of virus-like particles, and a murine model was used to determine the capacity of the recombinant proteins and particles to induce protective immunity. All of the proteins induced humoral immunity as measured by an ELISA against whole virus. However, only the antisera from animals immunized with VP4 neutralized virus and inhibited haemagglutination. Challenge of neonates born to animals immunized with VP4 protein on assembled particles or in cell lysates showed protection against challenge with both homologous (bovine C486) and heterologous (SA-11) strains of rotavirus. In contrast, the offspring of mice immunized with VP6 were only partially protected. Neonates of animals immunized with virus-like particles composed of VP7 assembled on VP6 spherical particles were protected against challenge with the homotypic virus and significantly protected from a heterotypic challenge whereas unassembled VP7 protein provided only partial protection against challenge.

Animals↗
PubMed1993

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV.

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

Animals↗
PubMed1993

Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV.

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.

Animals↗
PubMed1992

Structural, functional, and immunological characterization of bovine herpesvirus-1 glycoprotein gl expressed by recombinant baculovirus.

The major glycoprotein complex gl of bovine herpesvirus-1 was expressed at high levels (36 micrograms per 1 x 10(6) cells) in insect cells using a recombinant baculovirus. The recombinant gl had an apparent molecular weight of 116 kDa and was partially cleaved to yield 63-kDa (glb) and 52-kDa (glc) subunits. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells, even though the cleavage sites of authentic and recombinant gl were shown to be identical. The oligosaccharide linkages were mostly endoglycosidase-H-sensitive, in contrast to those of authentic gl, which has mostly endoglycosidase-H-resistant linkages and an apparent molecular weight of 130/74/55 kDa. Despite the reduced cleavage and altered glycosylation, the recombinant glycoprotein was transported and expressed on the surface of infected insect cells. These surface molecules were biologically active as demonstrated by their ability to induce cell-cell fusion. Fusion was inhibited by three monoclonal antibodies specific for antigenic domains I and IV on gl. Domain I maps to the extracellular region of the carboxy terminal fragment glc and domain IV to the very amino terminus of the glb fragment, indicating that domains mapping in two distinct regions of gl function in cell fusion. Monoclonal antibodies specific for eight different epitopes recognized recombinant gl, indicating that the antigenic characteristics of the recombinant and authentic glycoproteins are similar. In addition, the recombinant gl was as immunogenic as the authentic gl, resulting in the induction of gl-specific antibodies in cattle.

Amino Acid Sequence↗
PubMed1992

Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus.

The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

Adenoviridae↗
PubMed1991

The S2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells.

The hemagglutinin/esterase (HE), spike precursor (S) and the S1 and S2 subunits of the spike precursor protein of bovine coronavirus were expressed in Spodoptera frugiperda (Sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. Extensive syncytia formation was observed in cells infected with the S2 recombinant but not with the HE or S1 recombinant baculoviruses. Fusion of Sf9 cells expressing the intact S protein precursor was evident after trypsin treatment. These results demonstrate that proteolytic cleavage of the S spike precursor is required for fusion induction and that the fusion is mediated by the S2 subunit. These observations may reflect the biological role of the S2 subunit in fusion-penetration during bovine coronavirus infection.

Amino Acid Sequence↗
PubMed1991

Structural analysis of the conformational domains involved in neutralization of bovine coronavirus using deletion mutants of the spike glycoprotein S1 subunit expressed by recombinant baculoviruses.

Two conformation-dependent neutralizing epitopes, A and B, have been mapped to the S1 subunit of the S spike glycoprotein of bovine coronavirus (BCV). In order to characterize the structure of these antigenic sites, we constructed a series of cDNA clones encoding deleted or truncated S1 derivatives and expressed the modified genes in insect cells using recombinant baculoviruses. Monoclonal antibodies directed against epitopes A and B recognized only the mutant S1 polypeptides containing amino acids 324-720, as demonstrated by immunoprecipitation and Western blot analysis in the absence of beta-mercaptoethanol. In addition, two domains within this region were identified and only mutants containing both domains were immunoreactive, indicating that both were critical in the formation of the antigenic determinants. One domain was localized between residues 324 and 403 and the other at residues 517-720. Deletion of either domain inhibited extracellular secretion of the mutant proteins whereas mutants containing both or none of the domains were secreted efficiently. This observation suggests a vital function of the native conformation of the S1 protein in both antigenic structure and intracellular transport. Antigenic determinants A and B were not distinguished, but these determinants appeared to require both domains for epitope formation. Our results suggest that the antigenic determinants formed by two domains are likely associated with the probable polymorphic region of the BCV S1 subunit.

Animals↗
PubMed1991

Bovine coronavirus nonstructural protein ns2 is a phosphoprotein.

To investigate the nature of the bovine coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a beta-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [32P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in coronavirus-infected cells.

Amino Acids↗