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Biomedical subjects

E R James

Publications and source records attributed to E R James.

At least 19 recordsLinked to original sources
PubMed2005

Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment.

Toxoplasma gondii affects a wide variety of hosts including threatened southern sea otters (Enhydra lutris nereis) which serve as sentinels for the detection of the parasite's transmission into marine ecosystems. Toxoplasmosis is a major cause of mortality and contributor to the slow rate of population recovery for southern sea otters in California. An updated seroprevalence analysis showed that 52% of 305 freshly dead, beachcast sea otters and 38% of 257 live sea otters sampled along the California coast from 1998 to 2004 were infected with T. gondii. Areas with high T. gondii exposure were predominantly sandy bays near urban centres with freshwater runoff. Genotypic characterisation of 15 new T. gondii isolates obtained from otters in 2004 identified only X alleles at B1 and SAG1. A total of 38/50 or 72% of all otter isolates so far examined have been infected with a Type X strain. Type X isolates were also obtained from a Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Molecular analysis using the C8 RAPD marker showed that the X isolates were more genetically heterogeneous than archetypal Type I, II and III genotypes of T. gondii. The origin and transmission of the Type X T. gondii genotype are not yet clear. Sea otters do not prey on known intermediate hosts for T. gondii and vertical transmission appears to play a minor role in maintaining infection in the populations. Therefore, the most likely source of infection is by infectious, environmentally resistant oocysts that are shed in the feces of felids and transported via freshwater runoff into the marine ecosystem. As nearshore predators, otters serve as sentinels of protozoal pathogen flow into the marine environment since they share the same environment and consume some of the same foods as humans. Investigation into the processes promoting T. gondii infections in sea otters will provide a better understanding of terrestrial parasite flow and the emergence of disease at the interface between wildlife, domestic animals and humans.

Animals↗
PubMed2004

An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality.

Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.

Animals↗
PubMed2001

Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

ATP Binding Cassette Transporter 1↗
PubMed2001

Identification of the first reported splice site mutation (IVS7-1G-->A) in the aminomethyltransferase (T-protein) gene (AMT) of the glycine cleavage complex in 3 unrelated families with nonketotic hyperglycinemia.

A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.

Amino Acid Oxidoreductases↗
PubMed2001

Recurrent mutations in P- and T-proteins of the glycine cleavage complex and a novel T-protein mutation (N145I): a strategy for the molecular investigation of patients with nonketotic hyperglycinemia (NKH).

Screening a DNA bank from 50 patients with enzymatic confirmation of their diagnosis of nonketotic hyperglycinemia gave allele frequencies of 5% for R515S of P-protein (glycine decarboxylase) and 7% for R320H of T-protein (aminomethyltransferase). In a previous report we found that 3% of the same patient alleles were positive for T-protein IVS7-1G>A. In total, testing for these three mutations identified 15% of alleles and positive results (one or two mutations) were found in 11 of the 50 patients. In addition, a novel point mutation in T-protein, N145I, was found in a single case and a PCR/restriction enzyme assay was developed for its detection.

Amino Acid Oxidoreductases↗
PubMed2001

Immunisation of mice with fractions derived from the intestines of Dirofilaria immitis.

Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.

Animals↗
PubMed2001

The antioxidant system beta-D(+) glucose-glucose oxidase-catalase: tests for pyrogenicity and antigenicity.

PURPOSE: beta-D(+) glucose-glucose oxidase (E.C.# 1.1.3.4)-catalase (E.C.# 1.11.1.6) (GO-CAT) is being investigated as a new antioxidant system for use in pharmaceutical solutions. This study reports the results of tests for pyrogenicity and antigenicity of GO-CAT derived from Aspergillus niger when used parenterally in autoclaved preparations. METHODS: The Limulus amebocyte lysate (LAL) method was used to test the pyrogenicity of native GO-CAT. Pyrogenicity/antigenicity was evaluated in vivo by injecting autoclaved GO-CAT into New Zealand white rabbits. Antigenicity was also evaluated by Ouchterlony and Western blotting. RESULTS: None of the native GO-CAT concentrations tested (up to 30.83 u/ml) produced a positive gel clot in the LAL test, thereby suggesting its non-pyrogenicity. The rabbits, which received seven injections of autoclaved GO-CAT over a period of eleven weeks, remained healthy during and after the GO-CAT injections. All Ouchterlony and Western blot assays using sera from rabbits injected with autoclaved GO-CAT were negative. Furthermore, autoclaved GO-CAT could not be detected in Ouchterlony assays using a mouse monoclonal antibody (GO40 mAb) to native A. niger glucose oxidase. Control samples containing native GO-CAT produced an antigen-antibody complex reaction in Ouchterlony assays against the GO40 mAb. Antigen-antibody complexes could be detected by non-denaturing PAGE in samples containing native GO-CAT/GO40 and boiled GO-CAT/GO40, but not in samples containing autoclaved GO-CAT/GO40. These results indicate autoclaved GO-CAT is neither pyrogenic nor antigenic. CONCLUSIONS: Based on these results, there is potential for the use of beta-D(+) glucose-glucose oxidase-catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.

Animals↗
PubMed2001

Inhibition of lens epithelial cell growth by induction of apoptosis: potential for prevention of posterior capsule opacification.

As a model of the cell proliferation occurring in posterior capsule opacification (PCO), lens epithelial cells (LEC) from human and rabbit capsulotomies, and a rabbit LEC line (N/N1003A) were grown in Dulbecco's Minimal Essential Media (MEM) with 10% fetal calf serum. LEC were exposed to the calcium ionophore, calcimycin, and viability was assessed by trypan blue staining, growth by 3H-thymidine incorporation and apoptosis by annexin/propidium iodide staining, calcein AM/ethidium bromide staining and DNA laddering. Human capsulotomy samples were similarly exposed to calcimycin, and apoptosis assayed by calcein AM/ethidium bromide staining. Calcimycin exposure induced apoptosis in both rabbit LEC cultures and human LEC, and changes leading to apoptosis could be detected within 30 minutes of calcimycin treatment. The decrease in viability and growth in human and rabbit LEC was dose-dependent. These data support the further evaluation of apoptosis induction as a possible treatment mechanism to prevent development of PCO following primary cataract surgery in humans.

Animals↗
PubMed2000

Biochemical and molecular investigations of patients with nonketotic hyperglycinemia.

The investigation of 14 unrelated patients with nonketotic hyperglycinemia led to the identification of mutations in 4 cases. Patients were initially categorized into probable P- or T-protein defects of the glycine cleavage enzyme complex, by the use of the glycine exchange assay without supplemental H-protein, then screened for mutations in the P-protein and T-protein genes, respectively.

Amino Acid Sequence↗
PubMed1998

Peroxidoxins: a new antioxidant family.

Peroxidoxins are a recently described family of antioxidants. They have an ancient origin, being present in organisms as primitive as the archaea, and they appear to be ubiquitous in living cells. Here, Sharon McGonigle, John Dalton and Eric James review the present understanding of the functions and mechanism of action of these enzymes and suggest that these antioxidants may represent the ;missing link' in the metabolism of reactive oxygen species by some protozoan and helminth parasites. Also, by performing sequence comparisons of homologues entered in the public databases, they have classified the parasite peroxidoxins as 1-cys or 2-cys enzymes. The discovery of these antioxidants may change our understanding of how reactive oxygen species, of parasite or host origin, are managed by parasites.

Journal Article↗
PubMed1996

Cryopreservation of an attenuated vaccine strain of the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that infects birds and mammals, including humans. T. gondii T-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure for T. gondii T-263 bradyzoites has been developed to meet the requirement for product stability. A Me2SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1 degree C min-1 to -40 degrees C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120 degrees C min-1) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation of T. gondii T-263 bradyzoites in individual vaccine doses.

Animals↗
PubMed1996

Onchocerca retinol- and ivermectin-binding protein activity.

The presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19.7 and 4.6 kDa, the 2 smaller M(r) peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19.7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin 4 peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate. However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all 4 fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19.7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19.7 kDa Onchocerca protein.

Animals↗
PubMed1994

Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga.

The ocular pathology associated with acanthamoebiasis may result, at least in part, from the excretory and secretory (E&S) products of the active Acanthamoeba trophozoites. To test this hypothesis, the ability of A. polyphaga (ATCC Strain 30461) trophozoite E&S products to digest collagen, the major constituent of the corneal stroma, was evaluated. The secreted proteinases of A. polyphaga were identified using in vitro azocoll degradation, activity-PAGE, radiolabeled extracellular matrix (ECM) degradation, and collagen degradation assays. Inhibitors of serine (phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate), cysteine [benzyloxyphenylalanyl-analyl fluoromethyl ketone, N-ethylmaleamide, ethylenediamine tetraacetic acid (EDTA), L-trans-3-carboxyiran-2-carbonyl-L-leucylagmatine], metallo- (1,10-phenanthroline, EDTA, phosphoramidon), and aspartyl (pepstatin A) proteinases were incorporated into the assays. Proteinase activity was detected in trophozoites and the E&S products of trophozoites but not in cysts. The azocoll and activity-PAGE assays indicated the presence of serine and cysteine proteinases, while degradation of the radiolabeled ECM by live trophozoites confirmed not only the presence of serine and cysteine proteinases but also metalloproteinase(s). Most proteinase activity occurred at neutral pH. Incubation of E&S with type I collagen did not yield the typical 3/4:1/4 products characteristic of vertebrate collagenases. These data suggest that E&S products of A. polyphaga contain multiple serine and cysteine proteinases with nonspecific collagenolytic activity and that metallproteinases form an additional minor constituent.

Acanthamoeba↗
PubMed1994

Superoxide dismutase.

Eric James here discusses the molecular forms of superoxide dismutase and looks at its potential role in the pathogenesis of parasitic infections.

Journal Article↗
PubMed1994

Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species.

Amino Acid Sequence↗
PubMed1993

Immunolocalization of superoxide dismutase in Dirofilaria immitis adult worms.

Superoxide dismutase (SOD) may not only perform a housekeeping role in filarial worms but also assist in defense against oxidants generated by host immune cells. Both Dirofilaria and Onchocerca adult filariae and microfilariae contain relatively high activities of the antioxidant enzyme SOD; adult Dirofilaria worms also secrete SOD in vitro. In addition, superoxide radicals are relatively impotent against Dirofilaria and Onchocerca microfilariae in vitro. In assessing the role of SOD, we determined the anatomic localization of SOD in D. immitis adult worms by immunolocalization at the light-microscopic level. We found that anti-D. immitis SOD did not stain parasite tissues homogeneously, in support of the hypothesis that SOD does not have only a housekeeping role and that the pattern of staining may suggest another role(s) for SOD.

Animals↗
PubMed1991

Dirofilaria immitis superoxide dismutase: purification and characterization.

Superoxide dismutase (SOD) was purified to apparent homogeneity from Dirofilaria immitis, the causative agent of Dog Heartworm disease which is prevalent in the Southeastern United States. The enzyme has a molecular weight of 18,000 under denaturing conditions with an isoelectric point of 5.6. Both values are similar to those found for previously purified helminth SODs. The amino acid analysis shows greater similarity with mammalian SODs than with the published Schistosoma mansoni SOD, probably because the S. mansoni enzyme appears to be an extracellular, not a cytosolic, SOD. Although SOD activity is easily detected in D. immitis homogenates, the hydrogen peroxide scavenging activities of catalase and glutathione peroxidase were below the limits of our assay. This suggests that D. immitis primary defense against oxidants may be SOD. We feel that this line of research may provide valuable insights into a vulnerable area of D. immitis that may be a good target for drug therapy.

Amino Acids↗