Biomedical subjects

R E Lyons

Publications and source records attributed to R E Lyons.

11 recordsLinked to original sources

PubMed2005

Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif.

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory-secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine 'KKXX' endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

Amino Acid Motifs↗

PubMed2004

Intestinal pathology during acute toxoplasmosis is IL-4 dependent and unrelated to parasite burden.

The role of interleukin-4 (IL-4) during the course of Toxoplasma gondii infection was studied using IL-4-/- mice and their wild-type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL-4 was demonstrated and IL-4-/- mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL-4-/- mice although conversely liver pathology was greater than in wild-type mice. Significantly, plasma IL-12 and IFN-gamma levels, which peaked at days 6 and 8, respectively, were higher in IL-4-/- mice. The exacerbatory role of IL-4 in the intestine was found by competitive RT-PCR not to be associated with increased parasite burdens but was related to comparative expression of IL-10.

Acute Disease↗

PubMed2001

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Amino Acid Sequence↗

PubMed2001

Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii.

The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, give rise to slowly dividing bradyzoites that reside in tissue cysts. Reactivation of disease is associated with conversion of bradyzoites to tachyzoites. A sensitive method for detection and assessment of the number of each life-cycle stage would be useful for following these events. Herein we describe the construction and validation of a plasmid (pSWITCH) containing a polycompetitor construct (SWITCH) for use in competitive reverse transcriptase-PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, which are exclusively expressed by tachyzoite and bradyzoite stages respectively, and for beta-tubulin, a gene expressed by both stages. Using cRT-PCR, samples can first be accurately normalized for expression of the housekeeping gene, beta-tubulin and then the relative levels of SAG2A and LDH2 expression compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonstrated here for the SAG2-related family of genes. This technique offers a powerful tool for studying the processes involved in tachyzoite and bradyzoite interconversion.

Animals↗

PubMed2001

Immunological studies of chronic ocular toxoplasmosis: up-regulation of major histocompatibility complex class I and transforming growth factor beta and a protective role for interleukin-6.

A murine model was used to characterize the local immune and inflammatory response during ocular toxoplasmosis. Major histocompatibility complex (MHC) class I, normally expressed at low levels in immune-privileged sites such as the eye, was up-regulated during infection as determined by competitive reverse transcriptase (RT)-PCR and immunocytochemistry for both beta2-microglobulin and the MHC class I heavy chain. However, the eyes of chronically infected mice also had increased levels of mRNA transcripts for transforming growth factor beta, a cytokine associated with immune privilege and constitutively expressed in normal eyes. Transcripts for a number of inflammatory mediators, including interleukin-6 (IL-6), were increased during chronic infection. The role of IL-6 was further investigated by comparing disease progression and the development of the local immune response in wild-type (WT) and IL-6-deficient mice (IL-6(-/-) mice). Following infection, IL-6(-/-) mice developed more severe inflammation in the retina and vitreous humor compared with WT mice. This increased severity of disease was associated with reduced ocular IL-1alpha and increased tumor necrosis factor alpha mRNA production compared with WT mice. Moreover, the increased severity of disease in IL-6(-/-) mice correlated with increased eye parasite burden as determined by RT-PCR for the Toxoplasma gondii bradyzoite-specific LDH2 gene. These results demonstrate alterations to components of immune privilege as a result of ocular toxoplasmosis and a role for IL-6 in controlling parasite numbers and inflammation in the eye.

Animals↗

PubMed1998

Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii.

This study compared the genes encoding cytoplasmic 70 kDa heat shock protein in virulent and avirulent strains of Toxoplasma gondii, to determine whether differences may contribute to the variation in protein expression levels previously reported for this protozoan parasite. A T. gondii PCR probe with homology to Eimeria acervulina cytoplasmic 70 kDa heat shock protein was used to screen a genomic DNA mini-library and isolate the gene from the virulent RH strain. The entire coding region was subsequently amplified from the avirulent ME49 strain by PCR. Alignment of the gene sequences revealed that the virulent RH strain had four copies of a seven-aa repeat unit (GGMPGGM) at the 3'- end of the gene compared with five copies in the avirulent ME49 strain. Comparison of this region among other virulent and avirulent strains revealed that this difference was consistent with virulence. Copy number estimation revealed that this gene is single-copy in both the RH and the ME49 strains. Analysis of mRNA expression revealed a 1.5- to 2-fold increase in transcription of this gene in virulent strains when compared with avirulent strains. For each strain, mRNA was observed at similar levels whether grown in vivo or in vitro. Also, heat-shock treatment of tachyzoites prior to harvest did increase mRNA levels in vitro. This suggests that post-transcriptional regulation of cytoplasmic 70 kDa heat shock protein may occur in T. gondii. Differences in cytoplasmic 70 kDa heat shock protein have been demonstrated at genomic and transcriptional levels in virulent strains compared with avirulent strains, suggesting that this 70 kDa heat shock protein may play an important role in the virulence of T. gondii.

Amino Acid Sequence↗

PubMed1995

Heat shock proteins of Toxoplasma gondii.

We have investigated heat shock protein (HSP) expression in mouse-virulent and -avirulent strains of Toxoplasma gondii by performing Western blot analysis using a monoclonal antibody against HSP65 of Mycobacterium bovis and a polyclonal antiserum against HSP70 of Plasmodium falciparum as primary antibodies. We initially observed that murine macrophages express HSP65 when infected with either virulent or avirulent strains, a result which contradicts previous reports. Differential HSP expression consistent which virulence was observed between strains, with high levels of a 70kDa HSP (HSP70) only detected in virulent strains in vivo. This protein was not observed in virulent strains in the immunocompromised mouse or in vitro, suggesting induction by immunological stress. This protein was only poorly expressed in avirulent strains. A 65kDa protein was observed in all strains in vivo and in vitro, suggesting a shared epitope with HSP70. These results are consistent with the hypothesis that the induced expression of HSP70 in virulent strains of T. gondii by immunological stresses may provide protection for these strains against cell damage associated with invasion of the host, allowing the virulent strains to persist as tachyzoites without the requirement for the encystation observed in avirulent strains.

Animals↗

PubMed1978

Comparative effectiveness of benzoyl peroxide and tretinoin in acne vulgaris.

A controlled, randomized clinical study comparing different commercially available benzoly peroxide gels and tretinoin cream was undertaken by four investigators using similar protocols and case report forms. Results were based on lesion-count reduction over an 8-week period. No significant difference in reduction of total acne lesions was detected between the benzoyl peroxide gels and tretinoin cream. However, benzoyl peroxide was significantly better (P less than .02) in reduction of papules and as good as tretinoin in reduction of comedones. Results of this study suggest that excessive drying and peeling are not essential in the reduction of acne lesions.

Acne Vulgaris↗

PubMed1978

Ultrastructural basis for the assay of topical acne treatments. Transmission and scanning electron microscopy of untreated comedones.

After two decades of using systemic antibiotics as a main treatment for acne, emphasis is again being placed on topical agents. Thus, it is highly desirable to have a procedure whereby the activity of the various compounds can be evaluated by direct visualization. Scanning electron microscopy combined with transmission electron microscopy provide the tools for such in assay. This study describes the ultrastructure of untreated comedones and provides the baseline or control data necessary for testing topical treatments. Comedones obtained by punch biopsies or comedo extractors were processed for electron microscopy and studied with an ETEC Autoscan Scanning Electron Microscope and with Philips EM 300 and EM 301 Transmission Electron Microscopes. Microorganisms, keratinized cells, sebum, and hairs interact with each other to form the comedonal mass. Corynebacterium acnes and Pityrosporum ovale proliferate abundantly in close association with sebum and penetrate the keratinized cells. Fine structural details of bacteria and yeasts as well as features of host-microbial relationship have been elucidated.

Acne Vulgaris↗

PubMed1977

A simplified technique for casting anatomical models.

For anyone desiring to cast anatomical models, a simplified technique for external casting has been developed using dental impression materials readily available in dental supply houses. These impressions yield life-like permanent models retaining topographical features of normal as well as pathological skin and nails.

Dental Impression Materials↗

PubMedDate not supplied

Response of multiple seizure types to corpus callosum section.

Twenty-four patients (16 men, 8 women) underwent corpus callosum section specifically for improvement of control of atonic or tonic seizures that resulted in falls and injuries. All patients suffered from multiple seizure types, including complex partial (CP) and tonic-clonic (TC) seizures, in addition to the tonic or atonic episodes. Preoperative seizure frequency was quantified for all types for 1 year immediately before surgery and for the most recent year since the procedure; average monthly counts were obtained for each seizure type. The period of follow-up since surgery averaged 43 months (range, 23-79 months). Statistically significant improvements were documented, not only for the atonic/tonic seizures (p less than 0.0001) for all patients, but also for TC seizures (17 patients; p less than 0.001) and CP seizures (20 patients; p less than 0.02). Six patients experienced an exacerbation of CP seizures postoperatively, and three developed new simple partial (SP) seizures. In all of the CP group and all three of the SP group, ictal video and EEG features suggested that the new seizures were an aborted expression of the previously generalized seizures. From these data, we conclude that callosotomy is an effective treatment for tonic, atonic, and TC seizures intractable to anticonvulsant medications. Three patients became seizure free. The procedure may also be useful for certain specific subgroups of CP epilepsy, but further studies are required before expanding callosotomy to intractable CP seizures not amenable to focal resection.

Accidental Falls↗