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J A Nelson

Publications and source records attributed to J A Nelson.

At least 109 records · Page 6Linked to original sources
PubMed1995

Interaction of manganese-mesoporphyrin with oleic acid vesicles.

We investigated the interaction between manganese(III)mesoporphyrin (MnMeso), a metalloporphyrin, and liposome membranes containing oleic acid (OA; cis-9-octadecenoic acid). MnMeso associates preferentially with OA but minimally with egg phosphatidylcholine (EPC). Using small unilamellar vesicles, we characterized the MnMeso-OA binding at neutral pH. Our data suggest that MnMeso binds to the OA bilayer with Kd = 6.8 x 10(-4) M; the binding stoichiometry of MnMeso-OA was 1:3.4. This OA-MnMeso interaction was analyzed further for changes in the T1 relaxation property of MnMeso. OA increased the T1 of MnMeso significantly more than did EPC, suggesting that the OA-MnMeso interaction was stronger than that of PC-MnMeso. The side-chain specificity of the OA interaction with this porphyrin derivative was further supported in an experiment with manganese mesotetra(4-sulfonatophenyl)porphine, which lacks hydrophobic side chains for OA interaction. The association of MnMeso with the OA membrane was proposed according to the structure of MnMeso and OA and further verified using electron microscopy. A strong association of MnMeso with OA, an absorption enhancer of the gastrointestinal tract, may be useful for delivery of MnMeso as an oral contrast agent for magnetic resonance imaging.

Animals↗
PubMed1995

Functional expression of the renal organic cation transporter and P-glycoprotein in Xenopus laevis oocytes.

The hypothesis that P-glycoprotein (P-gp) mediates the renal secretion of organic cations was tested by functional expression of mRNAs in the Xenopus laevis oocyte system. Efflux of 2'-deoxytubercidin (dTub), a substrate for the renal organic cation transporter (OCT) but not for P-gp, was enhanced by injection of renal mRNA but not by injection of mRNA from P-gp-overexpressing cells (MDCK cells transduced with the cDNA for human MDR1). The functional capacity of the MDCK-MDR mRNA was established by its ability to reduce the steady-state uptake of a classical P-gp substrate, vinblastine. Thus, these data indicate OCT and P-gp to be distinct entities. The Xenopus oocyte system provides a functional approach to further characterize the OCT.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia.

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.

Animals↗
PubMed1995

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine.

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 microCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [3H] deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [3H] deoxyadenosine selection, lymphoma cell clones resistant to 15 microCi/ml [3H] deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [3H] Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.

Animals↗
PubMed1995

Intravenous manganese-mesoporphyrin as a magnetic resonance imaging contrast agent: an experimental model using VX-2 carcinoma in rabbits.

RATIONALE AND OBJECTIVES: We investigated the potential of manganese (III) mesoporphyrin (Mn-mesoporphyrin) as a hepatobiliary contrast agent for magnetic resonance (MR) imaging in rabbits given VX-2 carcinoma liver implants. METHODS: Rabbits given VX-2 carcinoma liver implants (n = 8) were imaged before and after the intravenous (i.v.) administration of 0.04 mmol/kg Mn-mesoporphyrin. MR images were correlated with gross-specimen cross-sections. The distribution of Mn in various tissues following i.v. administration of 0.04 mmol/kg Mn-mesoporphyrin was determined using atomic absorption analysis. A standard panel of serum chemistries was followed over 7 days in six rabbits following this same dose of Mn-mesoporphyrin and compared with chemistries from two control rabbits. RESULTS: I.v. administration of 0.04 mmol/kg (25 mg/kg) Mn-mesoporphyrin resulted in improvement of tumor-to-liver contrast, with enhancement of normal liver (99.7 +/- 14.7%) and the gallbladder (442 +/- 116%), but not VX-2 tumor tissue (14.8 +/- 13.9%), (n = 8, p = .05). Analysis of tissue Mn levels 100 min after i.v. Mn-mesoporphyrin injection demonstrated preferential distribution of Mn to normal liver tissue (57.8 +/- 15.3 micrograms Mn/g) compared with VX-2 tumor (4.28 +/- 1.48 micrograms Mn/g). No significant change was found in the serum chemistries of six normal rabbits over a 7-day period after the i.v. administration of 0.04 mmol/kg Mn-mesoporphyrin. CONCLUSION: I.v. Mn-mesoporphyrin improved lesion-to-liver contrast because of preferential distribution of Mn-mesoporphyrin to normal liver parenchyma and bile.

Animals↗
PubMed1995

Two prototype blood-pool agents for contrast-enhanced magnetic resonance angiography of the portal vein in pigs.

RATIONALE AND OBJECTIVES: Macromolecular "blood-pool" agents such as polylysine-gadopentetate dimeglumine or albumin-gadopentetate dimeglumine, which provide prolonged intravascular enhancement, were tested as magnetic resonance (MR) angiography contrast agents for the portal vein in pigs. METHODS: Phase-contrast MR angiography of the portal veins was performed on six pigs before and after intravenous administration of polylysine-gadopentetate dimeglumine (n = 3) or albumin-gadopentetate dimeglumine (n = 3). RESULTS: The contrast-to-noise ratio of the portal veins was improved by 74% and 52%, respectively, using polylysine-gadopentetate dimeglumine or albumin-gadopentetate dimeglumine. More branches of the intrahepatic portal veins were visualized on postcontrast images. CONCLUSION: Blood-pool paramagnetic contrast agents improved the visualization of hepatic vasculature using phase-contrast MR angiography in our experimental model.

Albumins↗
PubMed1995

Liver contrast enhancement in primates using iopromide liposomes.

RATIONALE AND OBJECTIVES: We studied the feasibility of using iodinated liposomes as computed tomography (CT) liver contrast agents in nonhuman primates. METHODS: Iopromide-containing liposomes were investigated as reticuloendothelial (RES) contrast agents for CT scanning of the liver in normal adult baboons. For intravenous (i.v.) injection, liposomes were resuspended in mannitol solution, filtered under sterile conditions, and injected i.v. at doses of 200 and 400 mg l/kg to each of five anesthetized adult baboons. RESULTS: Animals tolerated the injections without measurable electrocardiographic changes and recovered uneventfully from anesthesia. Sequential CT scans of the baboons' upper abdomen acquired up to 60 min postinjection showed persistent enhancement of the liver 10-60 min after injection. Maximum enhancement levels were 36 and 61 delta Hounsfield units (delta H) after the 200- and 400-mg/kg doses, respectively. The mean time to plateau enhancement was 20 min with the 200-mg/kg dose and 10 min with the 400-mg/kg dose. The greatest splenic enhancements were 181 and 301 delta H after the 200- and 400-mg/kg doses, respectively. CONCLUSION: Iopromide liposomes are effective as RES contrast agents in primates.

Animals↗
PubMed1995

Magnetic resonance imaging of the hepatobiliary system: intestinal absorption studies of manganese mesoporphyrin.

RATIONALE AND OBJECTIVES: We studied the intestinal absorption of manganese mesoporphyrin (Mn-mesoporphyrin), a potential oral hepatobiliary contrast agent. METHODS: Mn-mesoporphyrin was complexed with monoolein and taurocholate (mixed micelles). Portal venous delivery and biliary excretion were measured after intestinal administration in rats and rabbits, and the mechanism of intestinal transport was studied in a combined lymph-bile fistula model in rats. T1-weighted magnetic resonance (MR) images of the liver were obtained in rats and domestic pigs before and after gastric administration of Mn-mesoporphyrin in mixed micelles. RESULTS: A 2.2-fold increase of portal venous Mn concentration was found 90 min after intestinal administration of the complex. None was found in the lymph collected from the thoracic duct, indicating a transcellular transport mechanism through the intestinal mucosa with portal venous delivery. Mn-mesoporphyrin levels in bile peaked between 240 and 270 min after administration (200-fold increase). The greatest liver enhancement (20-90%) was measured 360 min after administration. CONCLUSION: The feasibility of intestinal delivery of Mn-mesoporphyrin, a lipophilic hepatobiliary contrast agent was demonstrated.

Animals↗
PubMed1995

Serial magnetic resonance imaging of experimental atherosclerosis detects lesion fine structure, progression and complications in vivo.

A major problem in the study of lesions of atherosclerosis is the difficulty of imaging noninvasively the lesions and following their progression in vivo. To address this problem, we have developed advanced magnetic resonance techniques to noninvasively and serially image advanced lesions of atherosclerosis in the rabbit abdominal aorta. Both lumen and wall were imaged with high resolution. Progression of disease, resulting in increase in lesion mass, decrease in arterial lumen, or stenosis, and intralesion complications, can be detected. Images acquired in vivo correlate with the fine structure of the lesions of atherosclerosis, including the fibrous cap, necrotic core, and lesion fissures, as verified by gross examination, dissection microscopy, and histology. The ability to noninvasively identify the features of atherosclerotic plaques, has significant implications for determining risks and benefits associated with different therapeutic approaches.

Animals↗
PubMed1995

Persistence of Borrelia burgdorferi sensu lato in resolved erythema migrans lesions.

Erythema migrans skin lesions resulting from a tick bite and infection with Borrelia burgdorferi sensu lato eventually resolve, even without antibiotic therapy. The aim of the present study was to gauge the frequency of persistence of B. burgdorferi sensu lato in such lesions. Thus, the site of a previous lesion was biopsied and cultured in 48 patients: 39 with systemic or localized symptoms/signs and nine with no symptoms. B. burgdorferi sensu lato was isolated from biopsied skin from three symptomatic patients. Cultures of other tissues and fluids were negative. By genospecies-specific polymerase chain reaction (PCR) and pulsed-field gel electrophoretic (PFGE) analysis, two isolates were classified as Borrelia afzelii. The remaining isolate was a member of an unusual group of strains that type as B. burgdorferi sensu stricto by genospecies PCR but possess an atypical PFGE profile. All three patients had a dramatic clinical response to antibiotic treatment. These findings demonstrate the capacity of viable B. burgdorferi sensu lato organisms to persist in clinically normal-appearing skin at the site of a resolved erythema migrans rash for periods ranging from 2 months to 3.5 years. This observation may provide new insight into the organisms' ability to evade the host's immune response.

Adolescent↗
PubMed1995

Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.

Amino Acid Sequence↗
PubMed1995

Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription.

Three polypeptides are produced from the major immediate-early (IE) region of human cytomegalovirus by alternative splicing. The IE gene products regulate subsequent viral and cellular gene expression. We previously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransferase driven by the dihydrofolate reductase (DHFR) promoter and that an intact E2F site was required for the trans activation (M. Wade, T. F. Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mol. Cell. Biol. 12:4364-4374, 1992). With the availability of cDNA clones for the individual major IE proteins, we sought to determine which of these proteins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the 55- and 86-kDa major IE proteins from the IE2 region can each moderately trans activate the DHFR promoter and that the 72-kDa IE1 protein stimulates DHFR transcription to a much higher level. Furthermore, trans activation through the 72-kDa IE1 protein is in part E2F dependent, while activation by the 55- and 86-kDa IE proteins is E2F independent. We also demonstrate by in vitro pull-down assays that the 72-kDa IE1 protein can specifically interact with the DNA binding domain of E2F1 (amino acids 88 to 191) in the presence of nuclear extract. Moreover, antibodies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunoprecipitate IE72 from normal human fibroblast cells infected with human cytomegalovirus.

Base Sequence↗
PubMed1995

Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages.

Stimulation of monocytes/macrophages with activated nonadherent cells allows productive nonlytic growth of human cytomegalovirus (HCMV), but the viral replication cycle is delayed relative to replication of HCMV in human fibroblasts. Analysis of infected monocyte-derived macrophage (MDM) mRNA for major immediate-early (MIE 86, 72, and 55) and late (pp65 and gB) gene expression by reverse transcription PCR indicates that transcription peaks at 3 and 7 days postinfection (dpi), respectively. In contrast, in human fibroblast controls, mRNA for MIE and late gene expression peaked at 5 and 48 h postinfection, respectively. Consistent with reverse transcription PCR experiments, double-label antibody experiments first detected MIE antigen expression at 12 h postinfection, peaking at 3 dpi, and late (pp65 or gB) antigen expression at 5 dpi, peaking at 7 dpi. MIE antigen was not detected between 3 and 7 dpi but reappeared and was coexpressed with pp65 in enlarged MDM nuclei at 7 dpi. After 7 dpi, macrophages with numerous vacuoles containing large amounts of pp65 and gB were observed in culture. These vacuoles were frequently seen at cellular contact points, suggesting that cell-to-cell transfer of virus was the major mode of viral transmission. Consistent with this observation, infectious virus was recovered from MDM cellular lysates but not culture supernatant. The delayed growth and compartmentalization of HCMV in macrophages may allow the cell to accommodate the viral replication cycle without cell lysis. In addition, the macrophage may function as a vehicle for cell-to-cell transmission of HCMV.

Base Sequence↗
PubMed1995

Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

Amino Acid Sequence↗
PubMed1995

The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein.

A murine monoclonal antibody (I2) reacts strongly with the nucleus of human cytomegalovirus (HCMV)-infected human fibroblasts. Western blot (immunoblot) analysis using I2 demonstrated that a protein with an apparent molecular mass of 58-kDa (E58) was expressed at 5 h after infection, and levels increased through 72 h. Immunoblot screening of an early cDNA expression library resulted in a positive clone which hybridized to the right end of the XbaI C fragment of the HCMV Towne strain. Further analysis demonstrated that the E58-specific clone was homologous to the putative UL98 open reading frame, which has been proposed to encode the viral alkaline exonuclease homolog. RNA analysis demonstrated a 3.0-kb RNA which is expressed at early times after infection, as well as in the absence of viral DNA replication, and which is 3' coterminal with the pp28 (UL99) gene region. Insertion of the UL98 genomic sequence into a eucaryotic expression vector and subsequent Western blot analysis using I2 demonstrated that the expressed protein comigrated with E58 from infected cells. E58 also reacts specifically with a previously described antibody, anti-P2-1, which was proposed to recognize a putative late 58-kDa protein. E58 comigrates with the putative late 58-kDa protein, indicating that these two proteins are likely the same. Analysis of the UL98 promoter revealed a TATATAA sequence located at nucleotide 142525. Insertion of the putative promoter 5' to a reporter gene demonstrated that the UL98 promoter was activated in cotransfection experiments with IE1 and IE2 proteins. These studies demonstrate that UL98 is a bona fide early gene, which is consistent with its probable role as the viral alkaline exonuclease gene.

Amino Acid Sequence↗
PubMed1995

Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.

Animals↗